Dentin slice model of dental stem cells in a fibrin-agarose construct for dental pulp regeneration / Modelo de trozo de dentina de células madre dentales en una construcción de fibrina-agarosa para la regeneración de la pulpa dental

Objectives: To implement a dentin slice model of mesenchymal stem cells derived from dental tissues in a fibrin-agarose construct for dental pulp regeneration. Material and Methods: MSCs derived from different oral cavity tissues were combined with a fibrin-agarose construct at standard culture conditions. Cell viability and proliferation tests were assayed using a fluorescent cell dye Calcein/Am and WST-1 kit. The proliferation assay was evaluated at 24, 48, 72, and 96 hours. Also, we assessed the dental pulp stem cells (DPSCs) cell morphology inside the construct with histological stains such as Hematoxylin and Eosin, Masson's trichrome, and Periodic acid­Schiff. In addition, we elaborated a tooth dentin slice model using a culture of DPSC in the fibrin­agarose constructs co-adhered to dentin walls. Results: The fibrin-agarose construct was a biocompatible material for MSCs derived from dental tissues. It provided good conditions for MSCs' viability and proliferation. DPSCs proliferated better than the other MSCs, but the data did not show significant differences. The morphology of DPSCs inside the construct was like free cells. The dentin slice model was suitable for DPSCs in the fibrin-agarose construct. Conclusion: Our findings support the dentin slice model for future biological use of fibrin-agarose matrix in combination with DPSCs and their potential use in dental regeneration. The multipotency, high proliferation rates, and easy obtaining of the DPSCs make them an attractive source of MSCs for tissue regeneration.

Objetivos: Implementar un modelo de dentina con células madre mesenquimales derivadas de tejidos dentales en una constructo de fibrina-agarosa para la regeneración de la pulpa dental. Material y Métodos: Las MSC derivadas de diferentes tejidos de la cavidad oral se combinaron con una construcción de fibrina-agarosa en condiciones de cultivo estándar. Las pruebas de viabilidad y proliferación celular se ensayaron utilizando un kit de colorante celular fluorescente Calcein/Am y WST-1. El ensayo de proliferación se evaluó a las 24, 48, 72 y 96 horas. Además, evaluamos la morfología celular de las células madre de la pulpa dental (DPSC) dentro de la construcción con tinciones histológicas como hematoxilina y eosina, tricrómico de Masson y ácido peryódico de Schiff. Además, elaboramos un modelo de rebanadas de dentina dental utilizando un cultivo de DPSC en las construcciones de fibrina-agarosa coadheridas a las paredes de la dentina. Resultados: La construcción de fibrina-agarosa fue un material biocompatible para las MSC derivadas de tejidos dentales. Proporcionó buenas condiciones para la viabilidad y proliferación de las MSC. Las DPSC proliferaron mejor que las otras MSC, pero los datos no mostraron diferencias significativas. La morfología de las DPSC dentro de la construcción era como la de las células libres. El modelo de corte de dentina fue adecuado para DPSC en la construcción de fibrina-agarosa.Conclusión: Nuestros hallazgos respaldan el modelo de corte de dentina para el futuro uso biológico de la matriz de fibrina-agarosa en combinación con DPSC y su uso potencial en la regeneración dental. El multipotencial, las altas tasas de proliferación y la fácil obtención de las DPSC las convierten en una fuente atractiva de MSC para la regeneración de tejidos.

Construction of tissue engineered adipose by human adipose tissue derived extracellular vesicle combined with decellularized adipose tissues scaffold / 中国修复重建外科杂志

Objective: To explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles (hAT-EV) combined with decellularized adipose tissue (DAT) scaffolds, and to provide a new therapy for soft tissue defects. Methods: The adipose tissue voluntarily donated by the liposuction patient was divided into two parts, one of them was decellularized and observed by HE and Masson staining and scanning electron microscope (SEM). Immunohistochemical staining and Western blot detection for collagen type Ⅰ and Ⅳ and laminin were also employed. Another one was incubated with exosome-removed complete medium for 48 hours, then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation. The morphology of hAT-EV was observed by transmission electron microscopy; the nanoparticle tracking analyzer (NanoSight) was used to analyze the size distribution; Western blot was used to analyse membrane surface protein of hAT-EV. Adipose derived stem cells (ADSCs) were co-cultured with PKH26 fluorescently labeled hAT-EV, confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs. Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs co-cultured for 15 days. The DAT was scissored and then injected into the bilateral backs of 8 C57 mice (6-week-old). In experimental group, 0.2 mL hAT-EV was injected weekly, and 0.2 mL PBS was injected weekly in control group. After 12 weeks, the mice were sacrificed, and the new fat organisms on both sides were weighed. The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis in vivo. Results: After acellularization of adipose tissue, HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus; SEM showed that no cells and cell fragments were found in DAT, and thick fibrous collagen bundles could be seen; immunohistochemical staining and Western blot detection showed that collagen type Ⅰ and Ⅳ and laminin were retained in DAT. It was found that hAT-EV exhibited a spherical shape of double-layer envelope, with high expressions of CD63, apoptosis-inducible factor 6 interacting protein antibody, tumor susceptibility gene 101, and the particle size of 97.9% hAT-EV ranged from 32.67 nmto 220.20 nm with a peak at 91.28 nm. Confocal fluorescence microscopy and oil red O staining showed that hAT-EV was absorbed by ADSCs and induced adipogenic differentiation. In vivo experiments showed that the wet weight of fat new organisms in the experimental group was significantly higher than that in the control group ( t=2.278, P=0.048). HE staining showed that the structure of lipid droplets in the experimental group was more than that in the control group, and the collagen content in the control group was higher than that in the experimental group. The proportion of new fat in the experimental group was significantly higher than that in the control group ( t=4.648, P=0.017). Conclusion: DAT carrying hAT-EV can be used as a new method to induce adipose tissue regeneration and has a potential application prospect in the repair of soft tissue defects.

Treatment of Osteochondritis Dissecans of the Knee with Autologous Iliac Bone Graft and Hyaluronic Acid Scaffold

Osteochondritis dissecans (OCD) is a condition that corresponds to an idiopathic focal lesion affecting the subchondral bone with possible compromise of the stability of the adjacent cartilage. Treatment depends on the size of the lesion, cartilage stability, and the physeal status. The case reported is about an 18-year-old male patient who complained of suffering from knee pain for a period of ten months. Magnetic resonance imaging (MRI) revealed a lesion of 2 cm² in the medial femoral condyle that compromised the subchondral bone, compatible with OCD. He underwent surgery that consisted of filling the subchonral defect with an iliac crest autograft and sealing the defect with a hyaluronic acid scaffold. At the 12-month follow-up, the MRI shows complete healing and the patient has resumed sports activities. Management with autologous iliac crest graft and hyaluronic acid scaffold represents an effective alternative treatment for OCD.

Meniscal Transplants and Scaffolds: A Systematic Review of the Literature

The reported incidence of meniscal tears is approximately 61 per 100,000. In instances where preservation of the native meniscus is no longer a feasible option, meniscal allograft transplantation (MAT) and implants or scaffolds may be considered. The goal of this review was to compare the success and failure rates of two techniques, MAT and meniscal scaffolds, and make an inference which treatment is more preferable at the present time and future. Studies that met inclusion criteria were assessed for technique used, type of transplant used, number of procedures included in the study, mean age of patients, mean follow-up time, number of failures, failure rate, and reported reoperation rate. Fifteen studies for the MAT group and 7 studies for the meniscal scaffold group were identified. In this selection of studies, the average failure rate in the MAT group was 18.7% and average reoperation rate was 31.3%. The average failure rate in the meniscal scaffold group was 5.6%, and average reoperation rate was 6.9%. It appears that although MAT is associated with high reoperation and failure rates, the limited number of studies on both MAT and scaffolds and mainly short-term results of scaffold studies make it difficult to make an objective comparison.

Hydrogel and Platelet-Rich Plasma Combined Treatment to Accelerate Wound Healing in a Nude Mouse Model

BACKGROUND: Platelet-rich plasma (PRP) contains high concentrations of growth factors involved in wound healing. Hydrogel is a 3-dimensional, hydrophilic, high-molecular, reticular substance generally used as a dressing formulation to accelerate wound healing, and also used as a bio-applicable scaffold or vehicle. This study aimed to investigate the effects of PRP and hydrogel on wound healing, in combination and separately, in an animal wound model. METHODS: A total of 64 wounds, with 2 wounds on the back of each nude mouse, were classified into 4 groups: a control group, a hydrogel-only group, a PRP-only group, and a combined-treatment group. All mice were assessed for changes in wound size and photographed on scheduled dates. The number of blood vessels was measured in all specimens. Immunohistochemical staining was used for the analysis of vascular endothelial growth factor (VEGF) expression. RESULTS: Differences in the decrease and change in wound size in the combined-treatment group were more significant than those in the single-treatment groups on days 3, 5, 7, and 10. Analysis of the number of blood vessels through histological examination showed a pattern of increase over time that occurred in all groups, but the combined-treatment group exhibited the greatest increase on days 7 and 14. Immunohistochemical staining showed that VEGF expression in the combined-treatment group exhibited its highest value on day 7. CONCLUSIONS: This experiment demonstrated improved wound healing using a PRP–hydrogel combined treatment compared to either treatment individually, resulting in a decrease in wound size and a shortening of the healing period.

3D Printing Technology and Its Applications for Tissue/Organ Regeneration / 대한이식학회지

Three-dimensional (3D) printing, also known as additive manufacturing (AM), has been used frequently in regenerative or translational medicine. In addition, recent advances in 3D printing technologies have opened the door to 3D bio-printing, which uses cells, biocompatible materials, and scaffolding simultaneously to generate 3D functional tissues. Although tissue generation by bio-printing such as multilayered skin, bone, bladder, and vascular grafts has shown good results, there are still several challenges related to printing of entire organs, particularly modulation of vascular formation during organ regeneration. This article provides a background and introduction to bio-printing for creation of artificial organs and tissues.

Preparation and biomechanical property of genipin-crosslinked rat acellular spinal cord scaffolds / 中华创伤杂志

Objective To construct genipin-crosslinked rat acellular spinal cord scaffolds and evaluate their enzymatic degradation rate,biomechanical properties and cytotoxicity.Methods Rat spinal cord scaffolds were decellularized by chemical extraction and chemically crosslinked with 5 g/L genipin solution.Micro-structure of the uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were observed by HE staining and scanning electron microscopy and properties of pore size,porosity,water ratio,and degradation rate in 2.5 g/L trypsin enzyme solution were examined.Ultimate tensile strength and elastic modulus of normal rat thoracic spinal cord,uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were determined on Instron mechanical testing instrument.Rat bone marrow mesenchymal stem cells were cultured in lixivium of uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds and MTT assay for relative cell growth rate was test to evaluate the cytotoxicity of scaffolds.Results The uncrosslinked and the genipin-crosslinked acellular spinal cord scaffolds possessed a similar three-dimensional mesh-porous structure with a mean pore diameter about 30 μm and a porosity over 80％,but there was a statistical difference between the two groups(P ＞ 0.05).Water ratio of genipincrosslinked scaffolds was (229.7 ± 12.5) ％,far lower than (283.4 ± 11.2) ％ of uncrosslinked scaffolds (P ＜ O.05) ; genipin-crosslinked acellular spinal cord scaffolds had lower weight loss at each time point than the uncrosslinked acellular spinal cord scaffolds (P ＜ 0.05),but the stability in trypsin,ultimate tensile strength and elastic modulus of acellular spinal cord scaffolds were significantly enhanced by genipin-crosslinking (P ＜ 0.05).Furthermore,no obvious cytotoxicity was observed in the uncroslinked and genipin-crosslinked scaffolds.Conclusions Rat acellular spinal cord scaffolds present no obvious change in structure after genipin-crosslinking,but there is significant improvement in the biomechanical properties and ability against enzymatic degradation and no marked cytotoxicity.Hence,the genipincrosslinked scaffolds are promising in tissue engineering for spinal injury.