Conceived and designed the experiments: GP AG Rd MC GL AP. Performed the experiments: AG Rd MC GL. Analyzed the data: GP AG Rd AP MP. Contributed reagents/materials/analysis tools: MC AD. Wrote the paper: GP AG Rd MP.
The authors have declared that no competing interests exist.
Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear.
In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold.
In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future.
Engineering techniques capable of maximizing cell adhesion, performance and differentiation onto suitable scaffolds are needed to achieve and implement effective and efficient tissue reconstruction protocols. Polymer surface texturing and microstructures and physiological treatments with physical stimuli are found to ameliorate cell adhesion and differentiation and the overall tissue regeneration process
In any bioengineering application, the selection of the stem cell population to be used is a critical factor. This is because stem cells of different origin have distinct capabilities in terms of survival, differentiation potentials, responses to experimental manipulations, and ultimately performance and effectiveness in tissue reconstruction. In previous studies, we have found that stromal cells isolated from adult human dental pulp (SBP-DPSCs) are multipotential and can give rise to a variety of cell types and tissues including adipocytes, neural cell progenitors and myotubes
Numerous scaffold materials, compositions and designs have been experimented and compared over recent years
In bioengineering applications, bone formation is routinely monitored by a variety of histological, biochemical and molecular criteria. Chief among the latter is gene expression of growth factors
Vascular endothelial growth factor (VEGF)
The present study was thus conducted to test whether the osteogenic potentials of SBP-DPSCs would be affected by the physical characteristics of PLGA-based substrates. Given the presumed importance of microcavities, we prepared substrates that were either smooth or contained microcavities of different dimensions. We monitored a variety of cellular parameters over time as well as the histomorphometric characteristics of bone and production of BMP-2 and VEGF, two important bone-related growth factors.
As in previous studies from our laboratories, we isolated stromal stem cells from the dental pulps (SBP-DPSCs) of healthy individuals and sorted them to be c-Kit+/CD34+. After dental pulp extraction and digestion, almost 82.3±0.9% of cells obtained were viable.
The cells were grown under conditions favoring their commitment to the osteogenic lineage. By day 30 of culture the cells had become Runx-2 and CD44 double-positive by cytofluorimetric analysis as shown previously
Cells were then seeded onto PLGA 85:15 scaffolds. Scanning electron and confocal microscopy revealed that NMP (N-methyl-pyrrolidone)-treated scaffolds contained two types of concave microcavities: (i) large microcavities 80–120 µm in diameter and 40–100 µm in depth which we termed “primary” (
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Given their large dimensions, primary microcavities were able to fully contain cells as shown by scanning and confocal microscopy (
To determine whether cell-to-cell scaffold interactions modify phenotypic expression, SBP-DPSC cells were monitored for expression of alkaline phosphatase activity (ALP) at 24, 48, 72 and 96 hours after plating. ALP is an enzyme that is particularly important for osteogenic cells and is required for mineral deposition. Indeed, we observed that ALP was consistently greater in cells plated onto the microcavity-rich substrate (
The image shows the quantity of ALP during osteoblast differentiation at 24, 48, 72 and 96 hours within the cells cultured on the different surfaces. The data have been rounded to the closest integer value. The error bars are ±SD.*p<0.01. Each experiment was performed in triplicate (n = 3).
We then analyzed production and release of BMP-2 and VEGF, factors important for osteogenesis. Greater amounts of VEGF and BMP-2 were present in medium conditioned by cells grown on the microcavity-textured surface, and particularly at 72 and 96 hrs (
Each experiment was performed in triplicate (n = 3). The error bars are ±SD. *p<0.01. N indicates samples not NMP-treated; P indicates NMP-treated samples.
To test the possible influences of surface characteristics on bone formation
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Immunohistochemistry showed presence of mineralized extracellular matrix in day 60 microcavity-rich samples that contained bone markers including collagen I, BAP, OC and ON (
The panel shows positivity for Collagen I (
In this study we present evidence that dental pulp-derived stromal stem cells behave and respond differently when exposed to microcavity-rich versus smooth PLGA substrates. The concave texturing of the substrate elicits cytoarchitectural responses and adaptation in which the cells appear to favour intimate contacts with the secondary microcavities and cellular polarization. Such behaviour is accompanied by increased release of BMP-2 and VEGF into the culture medium and by higher ALP activity. It is likely that increased release of potent factors such as BMP-2 and VEGF and the higher ALP activity could have significant biological ramifications. By their proven involvement and potency in bone formation and angiogenesis, these factors and enzymatic activity may influence the responses and developmental program of stromal-derived cells via autocrine mechanisms and also influenced by surrounding cells via paracrine pathways. In this likely scenario, increased levels of BMP-2 and VEGF could be responsible for the greater amounts of bone tissue we observe after transplantation of the colonized microcavity-rich scaffold.
The coordinated increase in BMP-2 and VEGF levels suggest that a relationship exists between them during osteogenesis, as previously hypothesized
It is interesting to note that primary micro concavity diameters are similar to those seen in medullary trabecular spaces, while secondary micro concavities resemble niches in the stromal bone marrow, the regulatory depressions for hematopoiesis and ossification. It is conceivable that the structural features of the microcavity-rich surface may have somehow enabled the stromal cells to react more vigorously and favorably then the smooth surface. As stated above, cells on concave texturing are more polygonal in shape with phylopodia-like and lamellipodia-like extensions and appear to have a nuclear polarity that may represent an index of secretion and of other cellular activities, such as matrix formation. These parameters do not characterize the same cell populations plated onto smooth surfaces nor do they characterize gingival fibroblasts plated onto microcavity-rich substrate. It may then be that by mimicking
When a cell sits in a microcavity, when compared with the same cell laying on a flat surface, for the adhesion processes it recruits a higher number of integrins per volumetric unit
In conclusion, micro-concavities (subtractive pits texturing) elicit superior outcomes in terms of osteodifferentiation, cell maturation and specific protein production, leading to a neoformation of bone tissue of significant thickness. This information could be used to design new scaffolds for bone tissue engineering with textures capable of promoting more effective healing of bone and mineralization defects.
Human dental pulp was extracted from teeth of healthy adult subjects aged 20 to 45 years. Before extraction, each subject was checked for systemic and oral infection or diseases. Only disease-free subjects were selected for pulp collection. Each subject was pre-treated for a week with professional dental hygiene. Before extraction, the dental crown was covered with a 0.3% chlorexidin gel (Forhans, N.Y., USA), for 2 min. Dental pulp was gently removed by means of a dentinal excavator or a Gracey curette, immersed in a digestive solution containing penicillin 100 U/ml, streptomycin 100 µg/ml, 0.6 ml claritromycin 500 µg/ml, 3 mg/ml type I collagenase and 4 mg/ml dispase in PBS, and incubated for 1 h at 37°C. Following this incubation, the digested tissue mixture was filtered through a 70 µm Falcon strainer (Becton & Dickinson, Franklin Lakes, NJ, USA) to obtain a single cell suspension.
After filtration, the cells were placed in α-MEM culture medium supplemented with 20% FCS, 100 µM 2P-ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all purchased from Invitrogen, San Giuliano Milanese, Milan, Italy) and placed in 75 cm2 flasks with filtered caps. Flasks were incubated at 37°C in humidified atmosphere containing a 5% CO2 and medium was changed twice a week. After cell confluence, the cells were subdivided into new flasks and passaged a total of 10 times. Human gingival fibroblasts were collected during periodontal surgery and cultured as above.
As previously specified
Briefly, cells were detached using 0.02% EDTA in PBS and pelleted (10 min at 1,000 rpm), washed in 0.1% BSA in 0.1 M PBS at 4°C and incubated in a solution of 1 µl antibody/9 µl 0.1% BSA in 0.1 M PBS. Cells were washed in the same solution once and were processed for sorting (FACsorter, Becton & Dickinson, Franklin Lakes, NJ, USA). The mouse anti-human antibodies CD117 (c-kit), CD34, flk-1 and STRO-1 were from Dr. Torok-Storb through DBA, Segrate, Milan, Italy.
Osteogenic differentiation was achieved as reported by Laino et al.
To monitor differentiation, the cells were examined using mouse anti-human antibodies to CD44, the transcription factor RUNX-2 (all from Santa Cruz, CA, USA), type I and II BMP receptors (BMPr I–II) and VEGF receptor type II (Flk-1). For RUNX-2 analysis, cells were fixed in 4% paraformaldehyde in 0.1 M PBS and 0.2% Triton X-100 for 30 min at 4°C, washed twice in 0.1% BSA in 1M PBS and then incubated with RUNX-2 antibody.
To obtain scaffold with two surface texturing, PLGA 85:15 was carved as membranes (INION, Finland). (A) a smooth surface was obtained using PLGA membrane as sold by the manufacturer; and (B) a concave texturing was obtained treating the membrane with NMP, a chemical agent (by the same manufacturer) that is able to create controlled subtractive pits on the surface following manufacturer's instructions.
Aliquots of 100,000 SBP-DPSCs (CD44+/RUNX-2+) were gently plated on the scaffolds and cultured for 96 h in order to observe cell-substrate adhesion and protein expression. Gingival fibroblasts were used as control for each substrate. Plating efficiency assays were performed counting free-floating cells 12 hours after plating.
After 96 h scaffolds and cultured cells were transplanted into animals. Experiments were repeated at least four times.
After 96 h of culture, cells were fixed in 2.5% glutaraldehyde (EM grade) in 0.1 M phosphate buffer, postfixed in 0.1% OsO4 in the same buffered solution for 1 h and, after critical point drying and gold-palladium coating, were observed under a Scanning Electron Microscope (JEOL-6700F, Tokyo, Japan).
After 96 h of culture, cells were fixed for 15 min at room temperature using 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in 0.1 PBS, washed twice with 0.1 M PBS for 10 min at room temperature and incubated for 30 min with phalloidin-FITC (Sigma, Milan, Italy), then washed with 0.1 M PBS for 10 min and stained for 2 min with Hoechst Blue (Sigma, Milan, Italy). Cells were washed twice with 0.1 M PBS and then observed on a Zeiss LSM510 confocal microscopy (Carl Zeiss, Göttingen, Germany).
For alkaline phosphatase (ALP), samples of 100,000 differentiated SBP-DPSCs (CD44+/RUNX-2+) were detached after 24, 48, 72, 96 hours by treatment with PBS/EDTA (0.02%) and centrifuged for 10 min at 140 × g. The pellet was incubated with 1 ml of BMPurple solution (Roche, Segrate, Milan, Italy) for 8 hours in the dark. Supernatant absorbance was measured at 615 nm using a spectrophotometer. As a control, human ginigival fibroblasts cultured on scaffolds were used. Values are expressed as the ratio between sample and BMPurple stock solution. BMPurple solvent was used as blank.
In order to evaluate BMP-2 and VEGF levels within the cell layer, after 48, 72, 96 hours from plating onto scaffolds, all the cell layer (4×106 cells/sample) of SBP-DPSCs (CD44+/RUNX-2+) were lysed in RIPA buffer (1 mM EDTA, 50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% Triton X-100, 0.25% Na-deoxycholate, 1 mM sodium orthovanadate) with 1 µg/ml leupeptin (Sigma), 1 µg/ml pepstatin (Sigma), 1 µg/ml aprotinin (Sigma), and 1 mM PMSF (Sigma). Samples were centrifuged (16,000 × g at 4°C for 20 minutes) and supernatant was precleared on an orbital shaker for 1 h at 4°C with protein-A acrylic beads (Sigma). Following centrifugation (5 min at 12,000 g), protein contents of the supernatants were determined using the Bradford reagent (Bio-Rad, Milan Italy) at 595 nm. Aliquots of 0.5 ml were collected from each sample and analyzed with an ELISA kit for BMP2 or anti-VEGF (R&D, Milan, Italy).
In order to evaluate BMP-2 and VEGF levels in the culture medium, the complete supernatant medium was collected from cultures after 24, 48, 72 and 96 hours from plating (4×106 cells/sample) SBP-DPSCs onto scaffolds. After centrifugation to remove particulates, aliquots of 2 ml were stored at −20°C. After thawing at room temperature, 0.5 ml were collected from aliquots and analyzed with ELISA kit for BMP2 or anti-VEGF (R&D, Milan, Italy). As a control, human ginigival fibroblasts cultured on scaffolds were used.
PLGA membranes colonized with differentiated SBP-DPSCs (CD44+/RUNX-2+) (4×106 cells/sample) for 96 h were transplanted into the dorsal surface (i.e. subcutaneously) of 10–12 week Wistar rats (Charles River Laboratories Italia S.p.A., Calco, Lecco, Italy). Animals were immunocompromised using Cyclosporine A (Sandimmun, Novartis Pharma S.p.A., Origgio, Varese, Italy) at a dosage of 15 mg/kg body weight, administered 4 h before transplantation and then daily till sacrifice. During the two weeks following surgery, daily dosages were reduced gradually down to 6 mg/kg body weight. Transplants were recovered 30, 45 and 60 days after transplantation. All the above-mentioned procedures were approved by institutional small animal ethics committee.
Transplants were fixed in 4% paraformaldehyde in PBS for 48 h at 4°C pH 7.4, decalcified for 7 days in 10% EDTA in PBS at RT and then washed in PBS pH 7.4 at 4°C, dehydrated, embedded in paraffin and sectioned (5 µm thick). For histological analysis slides were deparaffinized, hydratated and stained with hematoxylin-eosin and Mallory staining. For immunofluorescence, sections were deparaffinized, hydratated, washed again in 0.1 M PBS, and then blocked in 3% FBS in 0.1 M PBS at room temperature. Mouse anti-human monoclonal antibodies were the following: bone alkaline phosphatase (BAP), osteonectin (US Biological, Swampscott, MA); anti-osteocalcin, anti-osteonectin, anti-BSP and anti- Collagen I (Santa Cruz, CA) were goat anti-human. The secondary antibodies were goat anti-mouse and mouse anti-goat (both FITC conjugated, Santa Cruz). In addition, to evaluate cell distribution inside tissues, DAPI counterstaining was performed. Samples were observed under fluorescence microscopy (X41, Olympus Optical Co. Europe, Hamburg, Germany). The specificity of each antibody was assessed by reacting the above mentioned antibodies with human bone samples from a mandible. Isotype-matched antibodies were used at the same concentrations as negative controls.
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