Spinal cord injury (SCI) is a complex pathological process. Based on the encouraging results of preclinical experiments, some stem cell therapies have been translated into clinical practice. Mesenchymal stem cells (MSCs) have become one of the most important seed cells in the treatment of SCI due to their abundant sources, strong proliferation ability and low immunogenicity. However, the survival rate of MSCs transplanted to spinal cord injury is rather low, which hinders its further clinical application. In recent years, hydrogel materials have been widely used in tissue engineering because of their good biocompatibility and biodegradability. The treatment strategy of hydrogel combined with MSCs has made some progress in SCI repair. This review discusses the significance and the existing problems of MSCs in the repair of SCI. It also describes the research progress of hydrogel combined with MSCs in repairing SCI, and prospects its application in clinical research, aiming at providing reference and new ideas for future SCI treatment.
Objective To compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells (NPCs)and bone marrow mesenchymal stem cells (BMSCs) in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus. Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits (male or female, weighing 150-200 g). BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits (male or female, weighing 1.0-1.5 kg). The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β glycerophosphate, and hydroxyethyl cellulose. Then, NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus. The viabil ities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture. The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope (SEM) 1 week after compound culture. The histology and immunohistochemistry examination were performed. The expressions of aggrecan and collagen type II mRNA were analyzed by RT-PCR 3 weeks after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at37 (after 15 minutes) due to crossl inking reaction. Acridine orange/propidium iodide staining showed that the viabil ity rates of NPCs and BMSCs in chitosan hydrogel were above 90%. The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold, with extracellular matrix on their surfaces. The results of HE, safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix. RT-PCR results showed that the expressions of collagen type II and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel, and 0.713 ± 0.058 and 0.852 ± 0.076 in BMSCs culture with chitosan hydrogel; showing significant difference (P lt; 0.05). Conclusion The thermo-sensitive chitosan hydrogel has good cellular compatibil ity. BMSCs culture with chitosan hydrogel maintains better cell shape, prol iferation, and extracellular matrix biosynthesis than NPCs.
Methylcellulose is a semi-flexible cellulose ether derivative, whose hydrogels are thermosensitive and reversible, with good biocompatibility and adjustable function, and its application has attracted much attention in the biomedical field. In this paper, the application of methylcellulose-based thermo-sensitive hydrogels in biomedical field was reviewed. Based on the mechanism of gelation and influencing factors of methylcellulose, this paper focused on the recent advances in biomedical applications of methylcellulose-based hydrogels, including drug delivery, regenerative medicine, and other related fields. The current achievements in these fields were summarized in the form of lists in this paper to provide ideas and tendencies for future research. Finally, the future development of multifunctional methylcellulose-based hydrogel materials with improved performance was also discussed.
Objective To introduce an injectable andin situ gelling gelatin hydrogel, and to explore the possibility as a carrier for demineralized bone matrix (DBM) powder delivery. Methods First, thiolated gelatin was prepared and the thiol content was determined by Ellman method, and then the injectable andin situ gelling gelatin hydrogel (Gel) was formed by crosslinking of the thiolated gelatin and poly (ethylene oxide) diacrylate and the gelation time was determined by inverted method. Finally, the DBM-Gel composite was prepared by mixing Gel and DBM powder. The cytotoxicity was tested by live/dead staining and Alamar blue assay of the encapsulated cells in the DBM-Gel. Forin vitro cell induction, C2C12 cells were firstly incubated onto the surface of the DBM and then the composite was prepared. The experiment included two groups: DBM-Gel and DBM. The alkaline phosphatase (ALP) activity was determined at 1, 3, 5,and 7 days after culture.In vivo osteoinductivity was evaluated using ectopic bone formation model of nude rats. Histological observation and the ALP activity was measured in DBM-Gel and DBM groups at 4 weeks after implantation. Results The thiol content in the thiolated gelatin was (0.51±0.03) mmol/g determined by Ellman method. The gelation time of the hydrogel was (6±1) minutes. DBM powder can be mixed with the hydrogel and injected into the implantation site within the gelation time. The cells in the DBM-Gel exhibited spreading morphology and connected each other in part with increasing culture time. The viability of the cells was 95.4%±1.9%, 97.3%±1.3%, and 96.1%±1.6% at 1, 3, and 7 days after culture, respectively. The relative proliferation was 1.0±0.0, 1.1±0.1, 1.5±0.1, and 1.6±0.1 at 1, 3, 5, and 7 days after culture respectively.In vitro induction showed that the ALP activity of the DBM-Gel group was similar to that of the DBM group, showing no significant difference (P>0.05). With increasing culture time, the ALP activities in both groups increased gradually and the activity at 5 and 7 days was significantly higher than that at 1 and 3 days (P<0.05), while there was no significant difference between at 1 and 3 days, and between 5 and 7 days (P>0.05). At 4 weeks after implantationin vivo, new bone and cartilage were observed, but no bone marrow formation in DBM-Gel group; in DBM group, new bone, new cartilage, and bone marrow formation were observed. The histological osteoinduction scores of DBM-Gel and DBM groups were 4.0 and 4.5, respectively. The ALP activities of DBM-Gel and DBM groups were respectively (119.4±22.7) and (146.7±13.0) μmol/mg protein/min, showing no significant difference (t=–2.085,P=0.082). Conclusion The injectable andin situ gelling gelatin hydrogel for delivery of DBM is feasible.
Objective To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits. Methods TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups (n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties. Results CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs (P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation (P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group (P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences (P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased (P<0.05), while there was no significant difference in the number of cells and vascularity (P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group (P<0.05), but there was no significant difference compared to the CS group (P>0.05). Conclusion TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.
The chemical extraction method was used to prepare the rat uterine decellularized scaffolds, and to investigate the feasibility of preparing the extracellular matrix (ECM) hydrogel. The rat uterus were collected and extracted by 1%sodium dodecyl sulfate (SDS), 3% TritonX-100 and 4% sodium deoxycholate (SDC) in sequence. Scanning electron microscopy, histochemical staining and immunohistochemistry was used to assess the degree of decellularization of rat uterine scaffold. The prepared decellularized scaffold was digested with pepsin to obtain a uterine ECM hydrogel, and the protein content of ECM was determined by specific ELISA kit. Meanwhile, the mechanical characteristic of ECM hydrogel was measured. The results showed that the chemical extraction method can effectively remove the cells effectively in the rat uterine decellularized scaffold, with the ECM composition preserved completely. ECM hydrogel contains a large amount of ECM protein and shows a good stability, which provides a suitable supporting material for the reconstruction of endometrium in vitro.
ObjectiveTo prepare adipose-derived stem cells (ADSCs) and chitosan chloride (CSCl) gel complex to study the biocompatibility and the feasibility of repairing the wounds of deep partial thickness scald in rats. MethodsADSCs were prepared by enzymogen digestion and differential adherence method from the subcutaneous adipose tissue of SPF grade 6-week-old male Sprague Dawley (SD) rats. Temperature sensitive CSCl gel was prepared by mixing CSCl, β glycerol phosphate, and hydroxyethyl cellulose in 8∶2∶2.5 ratio. The proliferation of ADSCs was measured by cell counting kit 8 (CCK-8) assay and the survival of ADSCs was detected by the Live/Dead flurescent staining in vitro. A deep partial thickness burn animal model was made on the back of 72 SPF grade 6-week-old male SD rats by boiled water contact method and randomly divided into 3 groups (n=24). Group A was blank control group, group B was CSCl hydrogel group, group C was ADSCs/CSCl gel group. The wound closure rate at 3, 7, 14, 21 days was observed after operation. The number of inflammatory cells at 7 days and epidermal thickness at 21 days were observed by HE staining after operation. The angiogenesis at 7 days was evaluated by immunohistochemistry staining with CD31 expression. ResultsCSCl had a temperature sensitivity, at 4℃, the temperature-responsive hydrogel was liquid and became solid at 37℃. The CCK-8 assay and Live/Dead flurescent staining confirmed that ADSCs could grow and proliferate in the ADSCs/CSCl hydrogel complex. General observation showed the wound closure ratio in group C was superior to groups A and B after operation (P<0.05). HE staining showed that at 7 days after operation, the wound healing of the three groups entered fibrous proliferation stage. Collagen deposition and inflammatory cell infiltration were observed in the dermis of each group. The proportion of inflammatory cells in group C was significantly lower than that in groups A and B, and in group B than in group A (P<0.01). At 21 days after operation, the fibrous connective tissues of neoepithelium and dermis in groups B and C were arranged neatly, and fibroblasts and neocapillaries could be seen. In group A, neoepidermis could also be seen, but the fibrous connective tissues in dermis were arranged disorderly and sporadic capillaries could be seen. The thickness of neonatal epidermis in group C was significantly larger than that in groups A and B, and in group B than in group A (P<0.01). CD31 immunohistochemistry staining showed that the neovascularization could be seen in all groups. The number of neovascularization in group C was significantly higher than that in groups A and B, and in group B than in group A (P<0.05). ConclusionThe ADSCs/CSCl hydrogel complex has a good biocompatibility and possessed positive effects on promoting the deep partial thickness scald wound repairing in rats.
Objective
To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering.
Methods
The experiment was divided into two parts: ① BRL cells were cultured with 50, 100, and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10%FBS in groups A1, B1, and C1, and simple H-DMEM-medium served as a control (group D1); ② BRL cells were seeded on 1%, 2%, and 3% PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2, B2, and C2, and collagen type I gel served as a control (group D2). At 1, 3, and 5 days after culture, the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining. The cell vitality was tested by cell counting kit-8 (CCK-8) assay. And the relative expressions of albumin (ALB), cytokeratin 18 (CK18), and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR).
Results
The Live/Dead fluorescent staining showed the cells survived well in all groups. CCK-8 results displayed that the absorbance (A) value of group C1 was significantly higher than that of group D1 at 5 days after culture with PSISM-medium, and there was no significant difference between groups at other time points (P>0.05). After cultured with PSISM hydrogels, theA values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days (P<0.05), theA value of group A2 was significantly higher than that of groups B2 and C2 at 5 days (P<0.05), but there was no significant difference between groups at other time points (P>0.05). RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression of AFP mRNA significantly decreased in groups A1, B1, and C1 when compared with group D1 (P<0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 (P<0.05). The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2, B2, and C2 than group D2 (P<0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 (P<0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 (P<0.05), but no significant difference was found between other groups (P>0.05).
Conclusion
PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte. PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.
Since the release rate of protein in hydrogels is directly dependent upon the size of the protein and the hydrogel, how to deliver low molecular weight protein for prolonged periods has always been a problem. In this article, we present a usage of self-assembling peptide (P3) with the RGD epitope on its N terminus. The concentration of the released insulin-like growth factor 1 (IGF-1) was determined by UV-vis spectroscopy and the release kinetics suggested a notable reduction of the IGF-1 release rate. Cell entrapment experiments revealed that IGF-1 delivery by biotinylated nanofibers could promote the proliferation of the mouse chondrogenic ATDC5 cells when compared with cells embedded within nanofibers with untethered IGF-1.
Acute kidney injury is a worldwide public health issue, and its treatment and management strategies continue to advance. In addition to traditional kidney replacement therapy, research in recent years has been focused on whole organ engineering and biofabrication of kidney assistive devices and bioinjections for in-body regeneration. Hydrogel materials show great potential in renal tissue engineering because of their good biocompatibility, thermal stability and controllable biochemical and mechanical properties. This article reviews the application of various hydrogel materials in renal tissue engineering to promote kidney regeneration and discusses the characteristics and applications of natural hydrogels and synthetic hydrogels, which is expected to further promote their clinical applications.
