Poly Lactic-co-glycolic Acid Scaffolds Research Articles

Biocompatibility of two experimental scaffolds for regenerative endodontics.

ObjectivesThe biocompatibility of two experimental scaffolds for potential use in revascularization or pulp regeneration was evaluated.Materials and MethodsOne resilient lyophilized collagen scaffold (COLL), releasing metronidazole and clindamycin, was compared to an experimental injectable poly(lactic-co-glycolic) acid scaffold (PLGA), releasing clindamycin. Human dental pulp stem cells (hDPSCs) were seeded at densities of 1.0 × 104, 2.5 × 104, and 5.0 × 104. The cells were investigated by light microscopy (cell morphology), MTT assay (cell proliferation) and a cytokine (IL-8) ELISA test (biocompatibility).ResultsUnder microscope, the morphology of cells coincubated for 7 days with the scaffolds appeared healthy with COLL. Cells in contact with PLGA showed signs of degeneration and apoptosis. MTT assay showed that at 5.0 × 104 hDPSCs, COLL demonstrated significantly higher cell proliferation rates than cells in media only (control, p < 0.01) or cells co-incubated with PLGA (p < 0.01). In ELISA test, no significant differences were observed between cells with media only and COLL at 1, 3, and 6 days. Cells incubated with PLGA expressed significantly higher IL-8 than the control at all time points (p < 0.01) and compared to COLL after 1 and 3 days (p < 0.01).ConclusionsThe COLL showed superior biocompatibility and thus may be suitable for endodontic regeneration purposes.

The Adjuvant Effect Of PLGA Is Mediated Via Dendritic Cells In Vivo

Event Abstract Back to Event The Adjuvant Effect Of PLGA Is Mediated Via Dendritic Cells In Vivo Sangeetha Srinivasan1 and Julia E. Babensee1 1 Georgia Institute of Technology, Wallace H. Coulter Department of Biomedical Engineering, United States Introduction: The number of applications using biomaterials is rapidly growing due to the diversity of physical and chemical properties they can offer; specifically, devices comprised of materials such as poly lactic-co-glycolic acid (PLGA) have been approved by the Food and Drug Administration (FDA) for clinical use. We have previously shown that PLGA is distinct in its interaction with the adaptive immune system, in that it acts as an adjuvant to a model antigen such as ovalbumin (OVA) to boost antigen-specific immunity[1]. To enable researchers to design improved devices using PLGA, we are interested in understanding its mechanism by which it acted as an adjuvant in vivo. We hypothesized that PLGA’s adjuvant effect in vivo is primarily mediated by its effects on dendritic cells – the most potent antigen-presenting cells of the immune system – in an antigen-specific manner. Accordingly, we used a CD11c-DTR mouse model[2] in which CD11c+ cells (of which DCs are the largest population) were conditionally ablated by delivering diphtheria toxin. These mice received OVA-laden PLGA scaffolds as dorsal subcutaneous implants while also having received an intravenous injection with fluorescently label OVA-specific CD4+ T-cells. After allowing sufficient time for the interaction each of these components in vivo, we examined mice spleens for clonal expansion of the exogenously delivered T-cells. Our results clearly indicate that the OVA-specific T-cells in the DT-treated CD11c-DTR mice had failed to proliferate in comparison with non-DT treated controls due to the lack of sufficient DCs in vivo to drive antigen presentation, thereby supporting our original hypothesis that PLGA’s adjuvant effect is mediated via DCs. Materials and Methods: CD11c-DTR, OT-II and wild-type C57BL/6 mice of 6-8 weeks were purchased from Jackson labs and quarantined for one week prior to any treatment. CD4+ OVA-specific T-cells were isolated from OT-II mice mice and fluorescently labeled with carboxy fluorescein succinimidyl ester (CFSE) (Invitrogen) and injected in mice intravenously. Diphtheria toxin (Sigma) was supplied as 4ng/g[2]of mouse body weight via intravenous tail vein injections once every two days in order to sustain CD11c+ depletion over a 6-day period. PLGA scaffolds were prepared as described before[3]. Briefly, 75:25 pellets were dissolved in dichloromethane and cast into Teflon dishes mixed with dehydrated OVA (Sigma) and 125 – 250um NaCl salt crystals acting as porogens. The scaffolds thus made were washed, dried and cut into 8mm disks and implanted in the dorsal subcutaneous pockets in mice – one scaffold per mouse. Mice were subsequently sacrificed and their splenocytes were isolated and analyzed using flow cytometry. Results: The DC depletion experiment was conducted over a 7-day period with the appropriate treatments and days depicted on figure 1. Splenocytes from 3 mice groups – DTR mice with DT treatment, (control) DTR mice without DT treatment and (control) C57BL/6 wild-type with DT treatment (all with a PLGA/OVA implant– were stained with fluorescent antibodies against specific T-cell markers. CFSE incorporates in the nucleus of cells and is propagated in their daughter cells on cell division, thereby reducing in the extent of fluorescence emitted by the subsequent progeny[2]. Figure 2 shows the fluorescence intensity of the originally injected cells (shown in black) while that of any of its progeny (shown in blue). The lack of attenuation of CFSE fluorescence signal in DT-treated DTR mice (fig. 2 - bottom right panel) compared to the control groups (fig. 2 – top and bottom left panels) indicate that OVA-specific T-cells were unable to proliferate in DT-treated mice and thereby due to the lack of CD11c+ cells. National Institute of Health Grant

Adipose-Derived Stem Cells Decrease Bone Morphogenetic Protein Type 2-Induced Inflammation In Vivo

Recombinant human bone morphogenetic protein type 2 (rhBMP-2) has been used to promote bone regeneration. In contrast, some reports have suggested rhBMP-2 does not provide advantages over autogenous bone grafting owing to the undesirable postoperative symptoms of this growth factor. Because the undesirable symptoms of rhBMP-2 are usually promoted by inflammation, this study evaluated the in vivo effect of human adipose-derived stem cells (ASCs) incorporated into polylactic co-glycolic acid (PLGA) scaffolds in decreasing the inflammatory response induced by a low dose of rhBMP-2. PLGA scaffolds were characterized and loaded with rhBMP-2 1, 2.5, or 5 μg per scaffold (n = 6) and the in vitro released protein amounts were quantified at 7 hours and 1, 7, and 21 days after loading (n = 3). The muscle tissue of 6 beagles received the following treatments: PLGA, PLGA plus rhBMP-2 (2.5 μg), and PLGA plus rhBMP-2 plus ASCs (1 × 10(6) ASCs). The samples were evaluated 45 days after surgery. Statistical analyses were performed and the P value was set at .05. PLGA plus rhBMP-2 plus ASCs yielded the smallest number of inflammatory foci (P < .001) and giant cells (P < .001) and the largest number of angiogenesis sites (P < .001). Human ASCs administered in vivo into PLGA scaffolds with a low dose of rhBMP-2 decrease tissue inflammation and increase angiogenesis in muscular sites.

Electrospun aligned PLGA and PLGA/gelatin nanofibers embedded with silica nanoparticles for tissue engineering

Aligned poly lactic-co-glycolic acid (PLGA) and PLGA/gelatin nanofibrous scaffolds embedded with mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. The mean diameters of nanofibers were 641±24nm for the pure PLGA scaffolds vs 418±85nm and 267±58nm for the PLGA/10wt% MSNPs and the PLGA/gelatin/10wt% MSNPs scaffolds, respectively. The contact angle measurement results (102°±6.7 for the pure PLGA scaffold vs 81°±6.8 and 18°±8.7 for the PLGA/10wt% MSNPs and the PLGA/gelatin/10wt% MSNPs scaffolds, respectively) revealed enhanced hydrophilicity of scaffolds upon incorporation of gelatin and MSNPs. Besides, embedding the scaffolds with MSNPs resulted in improved tensile mechanical properties. Cultivation of PC12 cells on the scaffolds demonstrated that introduction of MSNPs into PLGA and PLGA/gelatin matrices leads to the improved cell attachment and proliferation as well as long cellular processes. DAPI staining results indicated that cell proliferations on the PLGA/10wt% MSNPs and the PLGA/gelatin/10wt% MSNPs scaffolds were strikingly (nearly 2.5 and 3 folds, respectively) higher than that on the aligned pure PLGA scaffolds. These results suggest superior properties of silica nanoparticles-incorporated PLGA/gelatin eletrospun nanofibrous scaffolds for the stem cell culture and tissue engineering applications.

Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function

The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGA-DC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair.

Osteoinduction of Umbilical Cord and Palate Periosteum–Derived Mesenchymal Stem Cells on Poly(Lactic-Co-Glycolic) Acid Nanomicrofibers

The need for tissue-engineered bone to treat complex craniofacial bone defects secondary to congenital anomalies, trauma, and cancer extirpation is sizeable. Traditional strategies for treatment have focused on autologous bone in younger patients and bone substitutes in older patients. However, the capacity for merging new technologies, including the creation of nanofiber and microfiber scaffolds with advances in natal sources of stem cells, is crucial to improving our treatment options. The advantages of using smaller diameter fibers for scaffolding are 2-fold: the similar fiber diameters mimic the in vivo extracellular matrix construct and smaller fibers also provide a dramatically increased surface area for cell-scaffold interactions. In this study, we compare the capacity for a polymer with Federal Drug Administration approval for use in humans, poly(lactic-co-glycolic) acid (PLGA) from Delta polymer, to support osteoinduction of mesenchymal stem cells (MSCs) harvested from the umbilical cord (UC) and palate periosteum (PP). Proliferation of both UC- and PP-derived MSCs was improved on PLGA scaffolds. The PLGA scaffolds promoted UC MSC differentiation (indicated by earlier gene expression and higher calcium deposition), but not in PP-derived MSCs. Umbilical cord-derived MSCs on the PLGA nanomicrofiber scaffolds have potential clinical utility in providing solutions for craniofacial bone defects, with the added benefit of earlier availability.

Combined effects of connective tissue growth factor-modified bone marrow-derived mesenchymal stem cells and NaOH-treated PLGA scaffolds on the repair of articular cartilage defect in rabbits.

In cartilage tissue engineering using stem cells, it is important to stimulate proliferation and control the differentiation of stem cells to specific lineages. Here we reported a combined technique for articular cartilage repair, consisting of bone marrow mesenchymal stem cells (BMMSCs) transfected with connective tissue growth factor (CTGF) gene and NaOH-treated poly(lactic-co-glycolic) acid (PLGA) scaffolds. In the present study, BMMSCs or CTGF-modified BMMSCs seeded on PLGA or NaOH-treated PLGA scaffolds were incubated in vitro and NaOH-treated PLGA significantly stimulated proliferation of BMMSCs, while CTGF gene transfer promoted chondrogenic differentiation. The effects of the composite on the repair of cartilage defects were evaluated in rabbit knee joints in vivo. Full-thickness cartilage defects (diameter: 5 mm; depth: 3 mm) were created unilaterally in the patellar groove. Defects were either left empty (n = 18) or implanted with BMMSCs/PLGA (n = 18), BMMSCs/NaOH-treated PLGA (n = 18), or CTGF-modified BMMSCs/NaOH-treated PLGA (n = 18). The defect area was examined grossly, histologically, and mechanically at 6, 12, and 24 weeks postoperatively. Implanted cells were tracked using adeno-LacZ labeling at 6 weeks after implantation. Overall, the CTGF-modified BMMSCs/NaOH-treated PLGA group showed successful hyaline-like cartilage regeneration similar to normal cartilage, which was superior to the other groups using gross examination, qualitative and quantitative histology, and mechanical assessment. The in vivo viability of the implanted cells was demonstrated by their retention for 6 weeks after implantation. These findings suggested that a combination of CTGF-modified BMMSCs and NaOH-treated PLGA may be an alternative treatment for large osteochondral defects in high-loading sites.

Effect of fiber orientation of collagen-based electrospun meshes on human fibroblasts for ligament tissue engineering applications.

Within the past two decades polylactic-co-glycolic acid (PLGA) has gained considerable attention as a biocompatible and biodegradable polymer that is suitable for tissue engineering and regenerative medicine. In this present study, we have investigated the potential of PLGA, collagen I (ColI), and polyurethane (PU) scaffolds for ligament tissue regeneration. Two different ratios of PLGA (50:50 and 85:15) were used to determine the effects on mechanical tensile properties and cell adhesion. The Young's modulus, tensile stress at yield, and ultimate tensile strain of PLGA(50:50)-ColI-PU scaffolds demonstrated similar tensile properties to that of ligaments found in the knee. Whereas, scaffolds composed of PLGA(85:15)-ColI-PU had lower tensile properties than that of ligaments. Furthermore, we investigated the effect of fiber orientation on mechanical properties and our results indicate that aligned fiber scaffolds demonstrate higher tensile properties than scaffolds with random fiber orientation. Also, human fibroblasts attached and proliferated with no need for additional surface modifications to the presented electrospun scaffolds in both categories. Collectively, our investigation demonstrates the effectiveness of electrospun PLGA scaffolds as a suitable candidate for regenerative medicine, capable of being manipulated and combined with other polymers to create three-dimensional microenvironments with adjustable tensile properties to mimic native tissues.

Osteogenic differentiation and angiogenesis with cocultured adipose-derived stromal cells and bone marrow stromal cells

The purpose of this study was to determine the influence of cocultured adipose-derived stromal cells (ASCs) in enhancing the osteogenic differentiation and angiogenesis of bone marrow stromal cells (BMSCs) as well as the underlying mechanism and the optimal ratio. Two in vitro coculture models, segregated cocultures using transwell and mixed cocultures, were employed to assess the indirect and direct effects of coculture respectively. Coculture was carried out for 14 days using 1 × 105 BMSCs and ASCs of variable number. BMSCs, ASCs, or both were seeded in PLGA scaffold and implanted in the subcutaneous tissue of 25 nude mice for in vivo analysis of angiogenesis. To evaluate the orthotopic bone formation, critical size calvarial defects were created on 20 mice, and implanted with hydroxyapatite/β-tricalcium phosphate granules plus BMSCs, ASCs, or both. From both transwell and mixed coculture model, 1 × 105 BMSCs cocultured with 0.5 × 105 ASCs showed significantly greater osteogenic differentiation and mineralization than BMSCs alone. The mixed ASC/BMSC coculture at or above a ratio of 0.5/1 showed increased secretion of vascular endothelial growth factor (VEGF), and induced effective tube formation from human umbilical vein endothelial cells, which were comparable to ASCs. Cytokine profiling assay and gene expression study showed elevated levels of angiogenic factors VEGF and CXCL1, osteogenic factor Wnt5a as well as transforming growth factor (TGF)-βR1 and SMAD3 from BMSCs when cocultured with ASCs. After 5 weeks of implantation, polylactic-co-glycolic acid (PLGA)-ASCs-BMSCs had a number of vascular structures comparable to PLGA-ASCs and significantly greater than PLGA-BMSCs. Calvarial defects treated with ceramic/BMSCs/ASCs had greater area of repair and better reconstitution of osseous structure than the defects treated with ceramic/ASCs or ceramic/BMSCs after 10 weeks. In conclusion, ASCs added to BMSCs promoted osteogenesis and angiogenesis at the optimal ASC/BMSC ratio of 0.5/1.

A Study of Surface Modification of Poly(lactic-co-glycolic) Acid Using Argon Ion Irradiation

The effect of Argon ion irradiation to the surface properties of poly(lactic-co-glycolic) acid (PLGA) was studied. A beam of 170 keV Argon ions was implanted at different fluencies (1 × 1012, 1 × 1013, 1 × 1014, and 1 × 1015 ions/cm2). X-ray photoelectron spectroscopy (XPS) was used to analyze the evolution of the bonding microstructure of PLGA due to irradiation. Surface morphology was monitored using atomic force microscopy (AFM). AFM analysis shows that film roughness increased to maximum at the dose of 1 × 1014 ions/cm2 where the formations of hillocks were also detected. Hydrophilicity of PLGA is important for their applications in biomedicine such as bioscaffolds. Hydrophilicity was monitored using water contact angle measurements for both unmodified and ion-modified PLGA. It was observed that hydrophilicity of PLGA changes with the ion irradiation. This demonstrates that ion irradiation can be an alternative approach to control hydrophilicity of PLGA. PLGA scaffolds modified with ion irradiation could therefore be more suitable for the biomedical applications.

The Graft of Autologous Adipose-Derived Stem Cells in the Corneal Stromal after Mechanic Damage

This study was designed to explore the feasibility of using autologous rabbit adipose derived stem cells (rASCs) as seed cells and polylactic-co-glycolic acid (PLGA) as a scaffold for repairing corneal stromal defects. rASCs isolated from rabbit nape adipose tissue were expanded and seeded on a PLGA scaffold to fabricate cell-scaffold constructs. After 1 week of cultivation in vitro, the cell-scaffold complexes were transplanted into corneal stromal defects in rabbits. In vivo, the autologous rASCs-PLGA constructed corneal stroma gradually became transparent without corneal neovascularization after 12 weeks. Hematoxylin and eosin staining and transmission electron microscopy examination revealed that their histological structure and collagen fibril distribution at 24 weeks after implantation were similar to native counterparts. As to the defect treated with PLGA alone, the stromal defects remained. And scar tissue was observed in the untreated-group. The implanted autologous ASCs survived up to 24 weeks post-transplantation and differentiated into functional keratocytes, as assessed by the expression of aldehyde-3-dehydrogenase1A1 (ALDH1A1) and cornea-specific proteoglycan keratocan. Our results revealed that autologous rASCs could be one of the cell sources for corneal stromal restoration in diseased corneas or for tissue engineering of a corneal equivalent.

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈86% of the defect area and ≈61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.

Short-term administration of small molecule phenamil induced a protracted osteogenic effect on osteoblast-like MC3T3-E1 cells

Sustained administration (21-day treatment) of the small molecule phenamil has been proposed as an alternative osteogenic factor when used in conjunction with a biodegradable scaffold for in vitro osteogenesis. While promising, the major issue associated with small molecules is non-specific cytotoxicity. The aim of this study was to minimize the side-effects from small-molecule drugs by reducing the frequency of administration. Toward this goal, we investigated whether a shorter phenamil treatment is sufficient to induce in vitro osteogenesis. We compared the effects of short-term (12 h) and continuous treatments of phenamil on osteoblastic differentiation and mineralization. Alkaline phosphatase (ALP) and osteopontin (OPN) activity were used as markers for osteoblastic differentiation. Measurement of the calcium content of the extracellular matrix was used as the hallmark for in vitro bone formation after 21 days of culture. Our findings revealed that both short and continuous phenamil treatment triggers osteoblastic differentiation and mineralization of MC3T3-E1 cells on a biodegradable polymeric scaffold composed of polylactic-co-glycolic acid (PLAGA) at the same time points. In addition, in order to fabricate a phenamil-loaded PLAGA scaffold, the small molecule phenamil was physically absorbed onto the surface of scaffolds and the bioactivity of the loaded scaffolds was evaluated. Furthermore, biochemical analysis indicated that short phenamil treatment of cells was accompanied by upregulation in protein expression of integrin α5, p125(FAK) and phosphorylation of CREB. These effects may contribute to the downstream signalling cascade necessary for osteogenesis, and such responses may account for our observed protracted osteogenic differentiation in vitro. Copyright © 2013 John Wiley & Sons, Ltd.

Effect of various blending ratios on the cell characteristics of PCL and PLGA scaffolds fabricated by polymer deposition system

Threedimensional (3D) scaffolds for tissue engineering have been described using a variety of molding and solid freeform fabrication (SFF) technologies. In this study, we developed a polymer deposition system for the fabrication of a scaffold similar to a bio-mimic tissue or organ. A 3D scaffold with a controllable pore size and high porosity can be implemented using this system. A precision 200 μm nozzle, thermostat with a maximum temperature of 250°C, and dispenser with a maximum pressure of 850 kPa play important roles in determining the deposition characteristics of molten polymer. Poly (ɛ-caprolactone) (PCL) and poly-lactic-co-glycolic acid (PLGA) were used to fabricate a biodegradable 3D scaffold. The dimensions of this scaffold were 25.0 × 10.0 × 4.0 mm. Scanning electron microscopy (SEM, Tescan VEGA II LMU, Czech) was used to acquire images of the 3D scaffold. Biodegradable synthetic polymers were fabricated into 3D scaffolds for tissue engineering using various blending ratios: PCL, blended PCL(75)/PLGA(25), blended PCL(50)/PLGA(50), blended PCL(25)/PLGA(75), and PLGA. In this research, the compressive strength and modulus of the fabricated 3D scaffolds were measured from the stress-strain curves. Moreover, a CCK8 assay and the growth of MC3T3-E1 cells were evaluated at 37°C in a 5% CO2 incubator.

NELL‐1 promotes cell adhesion and differentiation via integrinβ1

NELL-1 (Nel-like molecule-1) is a secreted osteogenic growth factor first identified in human craniosynostosis (CS) patients. NELL-1 protein has been observed to promote bone and cartilage differentiation and to suppress adipogenesis in both in vitro and in vivo models. Despite these findings, the cell surface receptors of NELL-1 have remained unknown. In this study, we observed for the first time that NELL-1 promotes cell adherence in multiple cell lines, including ST2, C3H10T1/2, M2-10B4, ATDC5, and MC3T3 cells. Additionally, we found that NELL-1 binds to extracellular Integrinβ1 and induces cell focal adhesion. By utilizing siRNA methods, we determined that NELL-1 cell surface binding and enhanced cell attachment were dependent on Integrinβ1 expression. Finally, we observed that pre-coating of culture dishes or PLGA (polylactic-co-glycolic acid) scaffold with NELL-1 resulted in a significant increase in both cell attachment and osteogenic differentiation. Our results identify for the first time a cell surface target of NELL-1, Integrinβ1, and elucidate new functions of NELL-1 in promoting cell adherence and osteogenic differentiation.

Tissue engineered tympanic membrane repairment materials with adipose-derived stem cells

To explore the morphology, proliferative activity and adhesion of adipose-derived stem cells (ASC) seeded in the polylactic-co-glycolic acid (PLGA) scaffold, and to provide experimental data for fabricating tissue engineered tympanic membrane repairment materials. Wistar rats were selected, and the ASC were isolated and co-cultured with the PLGA scaffold. The morphology and proliferative activity of ASC in the scaffold were examined by immunofluorescence of Vimentin and Ki67 respectively. All the immunofluorescence signals were analyzed by a confocal laser scan microscopy system FLUOVIEW FV1000. The adhesion of ASC to the PLGA scalfold was determined by scanning electron microscopy. Immunofluorescence of vimentin showed rats ASC displayed normal morphology and grew orderly in the PLGA scalfold. Immunofluorescence of Ki67 showed the normal active proliferation of ASC in the scaffolds. The Ki67 mean positive index (x(-) ± s) of the ASC in the scalfold was (8.21 ± 1.76)%, while in control group (cultured without PLGA scalfold) was (9.06 ± 1.95)%. There was no statistic significance between the two groups (t = 1.03, P = 0.30). Scanning electron microscopy demonstrated ASC adhered well to the PLGA scalfold, the pseudopodia of ASC could also be observed and the proliferative cell conjunction was tight. ASC has good biocompatible to the PLGA scaffold and could normally adhere and proliferate in PLGA scaffold.

Fabrication of pillared PLGA microvessel scaffold using femtosecond laser ablation

One of the persistent challenges confronting tissue engineering is the lack of intrinsic microvessels for the transportation of nutrients and metabolites. An artificial microvascular system could be a feasible solution to this problem. In this study, the femtosecond laser ablation technique was implemented for the fabrication of pillared microvessel scaffolds of polylactic-co-glycolic acid (PLGA). This novel scaffold facilitates implementation of the conventional cell seeding process. The progress of cell growth can be observed in vitro by optical microscopy. The problems of becoming milky or completely opaque with the conventional PLGA scaffold after cell seeding can be resolved. In this study, PLGA microvessel scaffolds consisting of 47 μm × 80 μm pillared branches were produced. Results of cell culturing of bovine endothelial cells demonstrate that the cells adhere well and grow to surround each branch of the proposed pillared microvessel networks.

Reconstruction of rat calvarial defects with human mesenchymal stem cells and osteoblast-like cells in poly-lactic-co-glycolic acid scaffolds

Human mesenchymal stem cells (hMSCs) can be used for xenogenic transplantation due to their low immunogenicity, high proliferation rate, and multi-differentiation potentials. Therefore, hMSCs are an ideal seeding source for tissue engineering. The present study evaluates the reconstruction effects of hMSCs and osteoblast-like cells differentiated from hMSCs in poly-lactic-co-glycolic acid (PLGA) scaffolds on the calvarial defect of rats. Two bilateral full-thickness defects (5mm in diameter) were created in the calvarium of nonimmunosuppressed Sprague-Dawley rats. The defects were filled by PLGA scaffolds with hMSCs (hMSC Construct) or with osteoblast-like cells differentiated from hMSCs (Osteoblast Construct). The defects without any graft (Blank Defect) or filled with PLGA scaffold without any cells (Blank Scaffold) were used as controls. Evaluation was performed using macroscopic view, histology and immunohistochemical analysis respectively at 10 and 20 weeks after transplantation. In addition, fluorescent carbocyanine CM-Dil was used to track the implanted cells in vivo during transplantation. The results showed that while both hMSC Construct and Osteoblast Construct led to an effective reconstruction of critical-size calvarial defects, the bone reconstruction potential of hMSC Construct was superior to that of Osteoblast Construct in non-autogenous applications. Our findings verify the feasibility of the use of xenogenic MSCs for tissue engineering and demonstrate that undifferentiated hMSCs are more suitable for bone reconstruction in xenotransplantation models.

Up-regulated α-actin expression is associated with cell adhesion ability in 3-D cultured myocytes subjected to mechanical stimulation

This study was aimed to investigate the alteration of alpha-actin in three-dimensionally (3-D) cultured myocytes under cyclic tensile stress loading. Myocytes were collected from neonatal SD rat's lateral pterygoid muscle for primary cell culture. The third-passage cells were implanted and 3-D cultured in poly lactic-co-glycolic acid (PLGA) scaffold, and then subjected to cyclic tensile stress (0.5 Hz, 2,000 microstrain) for 0, 2, 4, 8, 12, and 24 h through a four-point bending strain system. The alpha-actin mRNA was investigated by semi-quantitative RT-PCR. The alpha-actin protein expression was examined by immunofluorescent cytochemistry, laser confocal scanning microscopy (LCSM), and image analysis technology. The dynamic adhesion of myocytes to PLGA scaffolds was investigated by fluorescence microscope and the viability of the myocytes was measured by MTT assay. After mechanical loading, the alpha-actin mRNA increased at 2 h and then declined. The alpha-actin protein expression kept increased until peaked at 12 h, but declined at 24 h. The time course changing of alpha-actin protein expression parallelled with that of cell adhesion ability. It is concluded that alpha-actin expression is probably associated with cell adhesion ability in myocytes subjected to mechanical stimulation.

Comparison of the synthetic biodegradable polymers, polylactide (PLA), and polylactic-co-glycolic acid (PLGA) as scaffolds for artificial cartilage

Chondrocytes are easily de-differentiated when cultured in monolayer, and tissue-engineered cartilage can be generated by seeding chondrocytes onto three-dimensional porous synthetic biodegradable polymers. In this study, we investigated the biochemical and molecular aspects of chondrocytes in a monolayer-culture system and selected the optimal subculture passages based on their de-differentiation. We also compared two commonly used synthetic biodegradable polymers, polylactide (PLA), and polylactic-co-glycolic acid (PLGA), for their suitability as scaffolds for artificial cartilage. De-differentiated chondrocytes were observed after two passages. These results suggested that the first cell passage was optimal for seeding as only a few chondrocytes secreted extracellular matrix components to form homogeneously compact cartilage. Substantially increased glycosaminoglycan and total collagen levels revealed that PLGA scaffolds were a better option for inducing cartilage tissue formation compared to the PLA scaffolds. Histological and immunohistochemical results showed that chondrocytes seeded into PLGA retained their morphological phenotype to a greater extent than those seeded into PLA.