3D Hydrogel Scaffolds Research Articles

Human hepatocytes loaded in 3D bioprinting generate mini-liver

Because of an increasing discrepancy between the number of potential liver graft recipients and the number of organs available, scientists are trying to create artificial liver to mimic normal liver function and therefore, to support the patient's liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering. We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups, with 15 mice in each group: control, hydrogel, hydrogel with L02 (cell line HL-7702), and hydrogel with hepatocyte growth factor (HGF). Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage (RILD). The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total bilirubin, CYP1A2, CYP2C9, glutathione S-transferase (a-GST), and UDP-glucuronosyl transferase (UGT-2) were compared among the groups. Hematoxylin-eosin (HE) staining and immunohistochemistry of cKit and cytokeratin 18 (CK18) of engrafted tissues were evaluated. The survival time of the mice was also compared among the four groups. 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT, AST, albumin, total bilirubin, CYP1A2, CYP2C9, a-GST and UGT-2 were significantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only (P<0.05). HE staining showed clear liver tissue and immunohistochemistry of cKit and CK18 were positive in the engrafted tissue. Mice treated with 3D scaffold+L02 cells had longer survival time compared with those in control and scaffold only (P<0.05). 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the reconstruction of liver tissues.

3D bioprinting of BM-MSCs-loaded ECM biomimetic hydrogels for in vitro neocartilage formation

In this work we demonstrate how to print 3D biomimetic hydrogel scaffolds for cartilage tissue engineering with high cell density (>107 cells ml−1), high cell viability (85 ÷ 90%) and high printing resolution (≈100 μm) through a two coaxial-needles system. The scaffolds were composed of modified biopolymers present in the extracellular matrix (ECM) of cartilage, namely gelatin methacrylamide (GelMA), chondroitin sulfate amino ethyl methacrylate (CS-AEMA) and hyaluronic acid methacrylate (HAMA). The polymers were used to prepare three photocurable bioinks with increasing degree of biomimicry: (i) GelMA, (ii) GelMA + CS-AEMA and (iii) GelMA + CS-AEMA + HAMA. Alginate was added to the bioinks as templating agent to form stable fibers during 3D printing. In all cases, bioink solutions were loaded with bone marrow-derived human mesenchymal stem cells (BM-MSCs). After printing, the samples were cultured in expansion (negative control) and chondrogenic media to evaluate the possible differentiating effect exerted by the biomimetic matrix or the synergistic effect of the matrix and chondrogenic supplements. After 7, 14, and 21 days, gene expression of the chondrogenic markers (COL2A1 and aggrecan), marker of osteogenesis (COL1A1) and marker of hypertrophy (COL10A1) were evaluated qualitatively by means of fluorescence immunocytochemistry and quantitatively by means of RT-qPCR. The observed enhanced viability and chondrogenic differentiation of BM-MSCs, as well as high robustness and accuracy of the employed deposition method, make the presented approach a valid candidate for advanced engineering of cartilage tissue.

Research on the printability of hydrogels in 3D bioprinting.

As the biocompatible materials, hydrogels have been widely used in three- dimensional (3D) bioprinting/organ printing to load cell for tissue engineering. It is important to precisely control hydrogels deposition during printing the mimic organ structures. However, the printability of hydrogels about printing parameters is seldom addressed. In this paper, we systemically investigated the printability of hydrogels from printing lines (one dimensional, 1D structures) to printing lattices/films (two dimensional, 2D structures) and printing 3D structures with a special attention to the accurate printing. After a series of experiments, we discovered the relationships between the important factors such as air pressure, feedrate, or even printing distance and the printing quality of the expected structures. Dumbbell shape was observed in the lattice structures printing due to the hydrogel diffuses at the intersection. Collapses and fusion of adjacent layer would result in the error accumulation at Z direction which was an important fact that could cause printing failure. Finally, we successfully demonstrated a 3D printing hydrogel scaffold through harmonize with all the parameters. The cell viability after printing was compared with the casting and the results showed that our bioprinting method almost had no extra damage to the cells.

Study of neuroprotective function of Ginkgo biloba extract (EGb761) derived-flavonoid monomers using a three-dimensional stem cell-derived neural model.

An in vitro three-dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell-derived neural co-culture model. Rat neural stem cells were differentiated into co-culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two-dimensional (2D) model using a two-step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase-3 activity in a dose-dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co-cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:735-744, 2016.

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds

BackgroundThree-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells.MethodsPrimary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer.ResultsWhen seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix.ConclusionsThese 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0677-5) contains supplementary material, which is available to authorized users.

Programming Mechanical and Physicochemical Properties of 3D Hydrogel Cellular Microcultures via Direct Ink Writing.

3D hydrogel scaffolds are widely used in cellular microcultures and tissue engineering. Using direct ink writing, microperiodic poly(2-hydroxyethyl-methacrylate) (pHEMA) scaffolds are created that are then printed, cured, and modified by absorbing 30 kDa protein poly-l-lysine (PLL) to render them biocompliant in model NIH/3T3 fibroblast and MC3T3-E1 preosteoblast cell cultures. Spatial light interference microscopy (SLIM) live cell imaging studies are carried out to quantify cellular motilities for each cell type, substrate, and surface treatment of interest. 3D scaffold mechanics is investigated using atomic force microscopy (AFM), while their absorption kinetics are determined by confocal fluorescence microscopy (CFM) for a series of hydrated hydrogel films prepared from prepolymers with different homopolymer-to-monomer (Mr ) ratios. The observations reveal that the inks with higher Mr values yield relatively more open-mesh gels due to a lower degree of entanglement. The biocompatibility of printed hydrogel scaffolds can be controlled by both PLL content and hydrogel mesh properties.

Automated quantitative assessment of three-dimensional bioprinted hydrogel scaffolds using optical coherence tomography.

Reconstructing and quantitatively assessing the internal architecture of opaque three-dimensional (3D) bioprinted hydrogel scaffolds is difficult but vital to the improvement of 3D bioprinting techniques and to the fabrication of functional engineered tissues. In this study, swept-source optical coherence tomography was applied to acquire high-resolution images of hydrogel scaffolds. Novel 3D gelatin/alginate hydrogel scaffolds with six different representative architectures were fabricated using our 3D bioprinting system. Both the scaffold material networks and the interconnected flow channel networks were reconstructed through volume rendering and binarisation processing to provide a 3D volumetric view. An image analysis algorithm was developed based on the automatic selection of the spatially-isolated region-of-interest. Via this algorithm, the spatially-resolved morphological parameters including pore size, pore shape, strut size, surface area, porosity, and interconnectivity were quantified precisely. Fabrication defects and differences between the designed and as-produced scaffolds were clearly identified in both 2D and 3D; the locations and dimensions of each of the fabrication defects were also defined. It concludes that this method will be a key tool for non-destructive and quantitative characterization, design optimisation and fabrication refinement of 3D bioprinted hydrogel scaffolds. Furthermore, this method enables investigation into the quantitative relationship between scaffold structure and biological outcome.

3D printing hydrogel and elastomer scaffolds in a fugitive support

3D printing hydrogel and elastomer scaffolds in a fugitive support

Micro-patterning directional fibronectin cues onto hydrogel scaffolds for cardiac tissue engineering

Event Abstract Back to Event Micro-patterning directional fibronectin cues onto hydrogel scaffolds for cardiac tissue engineering Quentin Jallerat1, Vinit S. Palayekar1, 2, Madeline N. Monroe1, 2, Ivan Batalov2, Gaurav Begur1, 2 and Adam W. Feinberg1, 2 1 Carnegie Mellon University, Biomedical Engineering, United States 2 Carnegie Mellon University, Materials Science And Engineering, United States Introduction: Myocardial infarction kills one in six people in the US, due to an inability to regenerate cardiac muscle lost after infarct. Cardiac muscle engineered in vitro is a potential approach to create new myocardium for infarct repair, however multiple challenges exist. Specifically, while spatially well-defined micropatterns of ECM proteins such as fibronectin (FN) have been shown to direct cardiomyocyte (CM) organization on 2D surfaces, extending this micropatterning to 3D scaffolds has been difficult. To address this, we have developed a technique to micropattern directional FN cues into 3D hydrogel scaffolds to direct CM organization. We compared CM alignment in response to FN lines on collagen type I (COL1) or fibrin hydrogels, with or without Matrigel and identified a specific scaffold composition that maximized CM uniaxial alignment. We then used this technique to build aligned 2D human CM sheets and multilayered 3D tissues. Methods: To engineer micropatterned hydrogel scaffolds, we modified a previously reported surface-initiated assembly technique[1]. Briefly, 20 μm wide FN lines were microcontact printed onto a sacrificial poly(N-isopropylacrylamide) (PIPAAm) substrate. PIPAAm was then dissolved to transfer the FN lines, first on gelatin, then onto hydrogels consisting of COL1 or fibrin with or without Matrigel. Embryonic chick CMs were cultured for 4 days onto patterned hydrogels, then fixed and stained for nuclei, actin, and α-actinin, with CMs seeded on micropatterned polydimethylsiloxane used as controls. Similarly, human embryonic stem cell derived CMs were seeded on COL1 patterned with FN lines. Orientational order parameter (OOP) of the actin cytoskeleton was analyzed to determine the hydrogel composition that maximized alignment of both CM types. To create multilayered constructs, engineered cardiac tissues were cultured for 2 days, stacked, and maintained in contact for 3 days before fixing. Results: FN patterns were well defined on the COL1 and fibrin, with <10% variation in line width. Chick CM alignment on fibrin was low for all concentrations of fibrin and Matrigel (OOP<0.5). In contrast, CMs aligned well on COL1 without Matrigel (OOP = 0.66 vs. 0.38 with Matrigel), and reached maximum anisotropy on high concentration COL1 (6 mg/mL) without Matrigel (OOP=0.75±0.1). Human CMs seeded on the high COL1 concentration substrate formed anisotropic contractile monolayers with distinct sarcomeres (OOP = 0.65±0.1). To build 3D tissues, we stacked cardiac sheets 3 layer high with a single layer thickness of 17 ± 1.8 µm. Conclusions: We engineered directional, microscale ECM cues into hydrogel scaffolds and successfully guided the alignment of cardiac cell sheets, including human CMs. The high concentration COL1 with integrated FN micropatterns maximized both chick and human CM alignment. Future work will focus on engineering multi-layered and aligned human cardiac tissues and assessing electrophysiology and contractility. Financial support from the Dowd-ICES fellowship to Q. Jallerat and from National Institutes of Health 1DP2HL117750 and American Heart Association 12SDG11800036 to A. W. Feinberg.

Ascorbic acid promotes extracellular matrix deposition while preserving valve interstitial cell quiescence within 3D hydrogel scaffolds.

Current options for aortic valve replacements are non-viable and thus lack the ability to grow and remodel, which can be problematic for paediatric applications. Toward the development of living valve substitutes that can grow and remodel, porcine aortic valve interstitial cells (VICs) were isolated and encapsulated within proteolytically degradable and cell-adhesive poly(ethylene glycol) (PEG) hydrogels, in an effort to study their phenotypes and functions. The results showed that encapsulated VICs maintained high viability and proliferated within the hydrogels. The VICs actively remodelled the hydrogels via secretion of matrix metalloproteinase-2 (MMP-2) and deposition of new extracellular matrix (ECM) components, including collagens I and III. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from an activated myofibroblastic phenotype to a quiescent, unactivated phenotype, evidenced by the loss of α-smooth muscle actin expression upon encapsulation. In an effort to promote VIC-mediated ECM production, ascorbic acid (AA) was supplemented in the medium to investigate its effects on VIC function and phenotype. AA treatment enhanced VIC spreading and proliferation, and inhibited apoptosis. AA treatment also promoted VIC-mediated ECM remodelling by increasing MMP-2 activity and depositing collagens I and III. AA treatment did not significantly influence the expression of α-smooth muscle actin (myofibroblast activation marker) and alkaline phosphatase (osteogenic differentiation marker). No calcification or nodule formation was observed within the cell-laden hydrogels, with or without AA treatment. These results suggest the potential of this system and the beneficial effect of AA in heart valve tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

3D printing scaffolds with hydrogel materials for biomedical applications

3D printing has now been recognized as a very practical technique to create 3D structures with milli-/micron-scale resolution. In tissue engineering, particularly, people utilize 3D printing technique to integrate biodegradable polymers to tissue scaffolds. Hydrogel is highly potential material that provides aqua environment and enables nutrition and oxygen transportation, all of which are requirements for cells. A combination of hydrogel and 3D printing makes the developed platforms more biocompatible, being a benign niche for cells culture and study both in vitro and in vivo. Therefore, this review briefly introduces the background of 3D printing and the status of 3D printing and hydrogel scaffolds for application in biomedical research.

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

HAp granules encapsulated oxidized alginate–gelatin–biphasic calcium phosphate hydrogel for bone regeneration

Bone repair in the critical size defect zone using 3D hydrogel scaffold is still a challenge in tissue engineering field. A novel type of hydrogel scaffold combining ceramic and polymer materials, therefore, was fabricated to meet this challenge. In this study, oxidized alginate–gelatin–biphasic calcium phosphate (OxAlg–Gel–BCP) and spherical hydroxyapatite (HAp) granules encapsulated OxAlg–Gel–BCP hydrogel complex were fabricated using freeze-drying method. Detailed morphological and material characterizations of OxAlg–Gel–BCP hydrogel (OGB00), 25wt% and 35wt% granules encapsulated hydrogel (OGB25 and OGB35) were carried out for micro-structure, porosity, chemical constituents, and compressive stress analysis. Cell viability, cell attachment, proliferation and differentiation behavior of rat bone marrow-derived stem cell (BMSC) on OGB00, OGB25 and OGB35 scaffolds were confirmed by MTT assay, Live–Dead assay, and confocal imaging in vitro experiments. Finally, OGB00 and OGB25 hydrogel scaffolds were implanted in the critical size defect of rabbit femoral chondyle for 4 and 8 weeks. The micro-CT analysis and histological studies conducted by H&E and Masson's trichrome demonstrated that a significantly higher (***p<0.001) and earlier bone formation happened in case of 25% HAp granules encapsulated OxAlg–Gel–BCP hydrogel than in OxAlg–Gel–BCP complex alone. All results taken together, HAp granules encapsulated OxAlg–Gel–BCP system can be a promising 3D hydrogel scaffold for the healing of a critical bone defect.

Novel thiol- amine- and amino acid functional xylan derivatives synthesized by thiol–ene reaction

In the present work, novel thioether xylans were synthesized via a simple procedure using water as solvent. First, allyl groups were introduced on the backbone of xylan by etherification of allyl chloride in aqueous alkaline conditions at 40°C, providing degree of substitution (DS) values up to 0.49. On the second step, the allyl groups were reacted with thioacetic acid, cysteamine hydrochloride or cysteine providing novel thiol-, amine- or amino acid functionalized xylans. The presented modular approach offers broad possibilities for developing new polysaccharide based materials. The thioacetic acid - ene reaction is reported for the first time for polysaccharide modification, yielding a protected thiol that can be stored at atmospheric conditions and can be deprotected by simple hydrolysis just prior to use, providing a versatile water soluble polythiol. The free thiol-groups were utilized for hydrogel formation through thiol–thiol oxidative coupling, allowing good control over the hydrogel shape, such as 3D hydrogel scaffolds and cross-linked foams. Further, the thiol-containing xylan was used to modify filter paper surface by a simple dipping method, which provides a novel and convenient way for introducing thiol-functionality on paper surface.

Construction of Tumor Tissue Microarray on a Microfluidic Chip

A microfluidic approach for tumor tissue microarray construction was developed in this work. The 64 cell arrays were prepared by integrating cell introduction, 3D hydrogel scaffold formation and perfusion culture on the microfluidic chip. By using breast cancer MCF-7 cell as a model, cell viabilities, densities, proliferation rates and intracellular pH values were monitored throughout cell culture, and immunohistochemical staining was performed using frozen tissue sections. Breast cancer cells cultured on chip aggregated into tumor spheroids, showing a tissue structure similar to glandular tube. E-cadherin and Vinculin staining indicated the formation of cell-cell and cell-mesenchyme connections. Cancer cells kept proliferating within 15 days after seeding, and the cell viability and cell proliferation rate detected on the 15th day were 85% and 10%, respectively. Intracellular pH assay showed acidification in the cells, which was ascribed to the accumulation of acidic metabolic products caused by shortage of oxygen inside the tumor tissue. The developed microfluidic approach for tumor tissue microarray construction is simple and efficient for tumor research.

Dose-related effects of sericin on preadipocyte behavior within collagen/sericin hybrid scaffolds

This paper aims at demonstrating the biocompatibility of recently developed 3D hydrogel scaffolds containing the same amount of collagen (COLL) and variable concentrations of sericin (SS) in order to find the most suitable formula for adipose tissue engineering (ATE) applications. These scaffolds were obtained by COLL crosslinking with glutaraldehyde followed by freeze-drying and, subsequently, seeded with 3T3-L1 preadipocytes. Scanning electron microscopy studies revealed the scaffolds׳ architecture and cellular colonization. Also, in vitro biocompatibility of the developed scaffolds was evaluated by LDH and MTT assays and Live/Dead analysis of 3T3-L1 preadipocyte populating these 3D matrices. The best results in terms of cell survival and proliferation status were obtained in the case of the hybrid COLL scaffold containing 40% SS (COLL–SS4). Furthermore, the biological performance of the analyzed COLL-based hydrogels at 5- and 8- days post-seeding was found to decrease as follows: COLL–SS4>COLL–SS2>COLL>COLL–SS6. Consequently, our study highlights that hybrid scaffolds obtained by the addition of variable concentrations of SS to a constant COLL composition positively influences the behavior of 3T3-L1 cells with the exception of the COLL–SS6 matrix (60% SS). Altogether, the data obtained recommend SS as a component of COLL-based hydrogels providing them with features that may be useful in ATE applications.

3D printed hydrogel scaffolds for perfused liver culture

3D printed hydrogel scaffolds for perfused liver culture

A 3D nanofibrous hydrogel and collagen sponge scaffold promotes locomotor functional recovery, spinal repair, and neuronal regeneration after complete transection of the spinal cord in adult rats

Central nervous system neurons in adult mammals display limited regeneration after injury, and functional recovery is poor following complete transection (>4 mm gap) of a rat spinal cord. A novel combination scaffold composed of 3D nanofibrous hydrogel PuraMatrix and a honeycomb collagen sponge was used to promote spinal repair and locomotor functional recovery following complete transection of the spinal cord in rats. We transplanted this scaffold into 5 mm spinal cord gaps and assessed spinal repair and functional recovery using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. The BBB score of the scaffold-transplanted group was significantly higher than that of the PBS-injected control group from 24 d to 4 months after the operation (P < 0.001–0.01), reaching 6.0 ± 0.75 (mean ± SEM) in the transplant and 0.70 ± 0.46 in the control groups. Neuronal regeneration and spinal repair were examined histologically using Pan Neuronal Marker, glial fibrillary acidic protein, growth-associated protein 43, and DAPI. The scaffolds were well integrated into the spinal cords, filling the 5 mm gaps with higher numbers of regenerated and migrated neurons, astrocytes, and other cells than in the control group. Mature and immature neurons and astrocytes in the scaffolds became colocalized and aligned longitudinally over >2 mm, suggesting their differentiation, maturation, and function. The spinal cord NF200 content of the transplant group, analyzed by western blot, was more than twice that of the control group, supporting the histological results. Transplantation of this novel scaffold promoted functional recovery, spinal repair, and neuronal regeneration.

In vivo bioluminescence imaging for viable human neural stem cells incorporated within in situ gelatin hydrogels.

BackgroundThree-dimensional (3D) hydrogel-based stem cell therapies contribute to enhanced therapeutic efficacy in treating diseases, and determining the optimal mechanical strength of the hydrogel in vivo is important for therapeutic success. We evaluated the proliferation of human neural stem cells incorporated within in situ-forming hydrogels and compared the effect of hydrogels with different elastic properties in cell/hydrogel-xenografted mice.MethodsThe gelatin-polyethylene glycol-tyramine (GPT) hydrogel was fabricated through enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2).ResultsThe F3-effluc encapsulated within a soft 1,800 pascal (Pa) hydrogel and stiff 5,800 Pa hydrogel proliferated vigorously in a 24-well plate until day 8. In vitro and in vivo kinetics of luciferase activity showed a slow time-to-peak after d-luciferin administration in the stiff hydrogel. When in vivo proliferation of F3-effluc was observed up to day 21 in both the hydrogel group and cell-only group, F3-effluc within the soft hydrogel proliferated more vigorously, compared to the cells within the stiff hydrogel. Ki-67-specific immunostaining revealed highly proliferative F3-effluc with compactly distributed cell population inside the 1,800 Pa or 5,800 Pa hydrogel.ConclusionsWe examined the in vivo effectiveness of different elastic types of hydrogels encapsulating viable neural stem cells by successfully monitoring the proliferation of implanted stem cells incorporated within a 3D hydrogel scaffold.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-014-0061-3) contains supplementary material, which is available to authorized users.