OBJECTIVE: To prepare chitosan-gelatin/hydroxyapatite (CS-Gel/HA) composite scaffolds, and to investigate the influence of components and preparing conditions to their micromorphology. METHODS: The CS-Gel/HA composite scaffolds were prepared by phase-separation method. Micromorphology and porosity were detected by using scanning electron microscope and liquid displacement method respectively. RESULTS: Porous CS-Gel/HA composite scaffolds could be prepared by phase-separation method, and their density and porosity could be controlled by adjusting components and quenching temperature. CONCLUSION: The study suggests the feasibility of using CS-Gel/HA composite scaffolds for the transplantation of autogenous osteoblasts to regenerate bone tissue.
Objective To review the recent advances in the application of graphene oxide (GO) for bone tissue engineering. Methods The latest literature at home and abroad on the GO used in the bone regeneration and repair was reviewed, including general properties of GO, degradation performance, biocompatibility, and application in bone tissue engineering. Results GO has an abundance of oxygen-containing functionalities, high surface area, and good biocompatibility. In addition, it can promote stem cell adhesion, proliferation, and differentiation. Moreover, GO has many advantages in the construction of new composite scaffolds and improvement of the performance of traditional scaffolds. Conclusion GO has been a hot topic in the field of bone tissue engineering due to its excellent physical and chemical properties. And many problems still need to be solved.
Objective To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. Methods Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge.The MSCs were embedded in acellular cartilage matrix. After 10 day’s combined culture, the specimens were observed with optical and electrical microscope.Results The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae.Conclusion The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.
Triply periodic minimal surface (TPMS) is widely used because it can be used to control the shape of porous scaffolds precisely by formula. In this paper, an I-wrapped package (I-WP) type porous scaffolds were constructed. The finite element method was used to study the relationship between the wall thickness and period, the morphology and mechanical properties of the scaffolds, as well as to study the compression and fluid properties. It was found that the porosity of I-WP type scaffolds with different wall thicknesses (0.1 ~ 0.2 mm) and periods (I-WP 1 ~ I-WP 5) ranged from 68.01% ~ 96.48%, and the equivalent elastic modulus ranged from 0.655 ~ 18.602 GPa; the stress distribution of the scaffolds tended to be uniform with the increase of periods and wall thicknesses; the equivalent elastic modulus of the I-WP type scaffolds was basically unchanged after the topology optimization, and the permeability was improved by 52.3%. In conclusion, for the I-WP type scaffolds, the period parameter can be adjusted first, then the wall thickness parameter can be controlled. Topology optimization can be combined to meet the design requirements. The I-WP scaffolds constructed in this paper have good mechanical properties and meet the requirements of repairing human bone tissue, which may provide a new choice for the design of artificial bone trabecular scaffolds.
Degenerative disc disease is a prevalent chronic disease that orthopaedic surgeons currently face as a difficulty. Tissue engineering represents the most promising possible therapeutic strategy for disc repair and regeneration. Surgery is the primary treatment for degenerative disc disease, but there are still inherent limits in practical practice. Electrospinning technique is a method for manufacturing nanoscale fibers with varied mechanical properties, porosity, and orientation, which can imitate the structural qualities and mechanical properties of natural intervertebral discs. Therefore, electrospinning materials can be utilized for disc regeneration and replacement. This article reviews recent advancements in disc tissue engineering and electrostatically spun nanomaterials typically utilized for the fabrication of disc scaffolds, as well as present and future techniques that may enhance the performance of electrostatically spun fibers.
Objective To prepare collagen-chitosan /nano-hydroxyapatite-collagen-polylactic acid (Col-CS/ nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1 ∶ 1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20 ∶ 1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.
ObjectiveTo review the research progress of tissue engineered scaffolds and stromal-derived factor 1 (SDF-1) composite graft. MethodsThe recent papers about SDF-1 with different kinds of tissue engineered scaffolds were reviewed and analyzed. The primary mechanism of SDF-1 homing function for stem cells was retrospected. The results of different kinds of tissue engineered scaffolds carrying SDF-1 for repairing the injured tissues and organs were reviewed. ResultsIt is shown that SDF-1 combined with tissue engineered scaffolds will play a role of multipotent stem cells chemotaxis, however, the exact chemotaxis mechanism has not been fully understood. It still needs more researches of SDF-1 effects in vivo. ConclusionAlthough some research progress has been made in regeneration in situ of tissue engineered scaffolds combined with SDF-1, it will need to further study on the mechanism of chemotactic functions of SDF-1 and its influence on proliferation and differentiation of cells.
Objective To compare the effect of two different methods of cell seeding on spatial distribution and gene expression of hBMSCs in biocoral scaffold in vitro cultures. Methods The composite of hBMSCs and biocoral scaffold was prepared by traditional seeding (group A) and fibrin glue seeding (group B). The seeding efficiency was measured after 30 minutes of incubation in group B and after 3 hours in group A. At 2, 7, 14 and 21 days after culture, the samples were harvestedand the serial longitudinal sections were cut for each embedded composite. The sections were stained with DAPI and were measured using fluorescence microscope with apotome under serial optical sections. The cell number in every 10 × objective field was automatically measured by AxioVision image analysis software and levels (from seeding surface to bottom L1-L5) or columns (from centre to margin) for comparing cell distribution were set up. The specific osteogenic genes [osteonectin (ON), core binding factor α1 (Cbfα1), osteocalcin (OC)] expression was measured by RT-PCR. Results The seeding efficiency was significantly higher in group B (88.32% ± 4.2%) than in group A (66.51% ± 12.33%, P lt; 0.01). At 2 days after culture, the cell number from L1 to L4 decreased gradully in two groups (P lt; 0.05); in the cell number of different columns, there was no significant difference in group A (Pgt; 0.05) whereas significant difference in group B (P lt; 0.05); there was no significant difference in gene expression between two groups (P gt; 0.05). At 7 days after culture, the cell number was less than that at 2 days in group A and there was significant difference among levels (P lt; 0.05). The cell number and osteogenic gene expression increased sharply and there appeared uniform cell distribution in group B (P gt; 0.05). The gene expression of ON and Cbfα1 in group B was higher than that in group A (Plt; 0.05). At 14 days after culture, the cell number in levels or columns in group A decreased sharply and was less than that at 7 days (P lt; 0.05); whereas the cell number was similar to that at 7 days in group B (P gt; 0.05). The OC gene expression reached the highest level in group B at 14 days. The gene expression was higher in group B than in group A (P lt; 0.05). At 21 days after culture, there was significant difference in the cell number among levels and in the gene expression between group A and group B (P lt; 0.05); there was no significant difference in the cell number among columns in two groups (Pgt; 0.05). In addition, the cell number of most levels and columns in group B was more than that in group A at 7, 14 and 21 days after culture (P lt; 0.05). Conclusion More uniform cell distribution with rapid prol iferation and osteogenic differentiation is available in different levels or columns of scaffold by fibrin glue seeding than by traditional seeding.
ObjectiveTo 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.MethodsThe 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.ResultsAfter 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).ConclusionDAT 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.
Objective To investigate the biocompatibility of p(3HB-co-3HH) and marrow mesenchymal stell cells (MSCs).Methods MSCs were inoculated to p(3HB-co-3HH), and then cultured for 2-4 weeks in vitro and embedded for 2 weeks in vivo. The growth, proliferation, morphology and phenotype properties of MSCs were observed by use of phase contrast microscope, electron microscope, HE staining and staining of type Ⅰ collagen. Results p(3HB-co-3HH) hadgood compatibility. The inoculated MSCs could be well-distributed, attached well and obtain the phenotype of MSCs in p(3HB-co-3HH). After osteogenic inducer were added, MSCs differentiated to osteoblasts and secreted matrix. Type Ⅰ collagen was stained positively by immunohistochemical techenique. Conclusion The above results demonstrate that there is satisfactory biocompatibility betweenp(3HB-co-3HH) and MSCs.