The attachment and growth behavior of osteoblast-like cells on microtextured surfaces

In previous studies, we showed that the application of microgrooves on a surface can direct cellular morphology and the deposition of mineralized matrix of osteoblast-like cells (Biomaterials 20 (1999) 1293; Clin. Oral Impl Res. 11 (2000) 325). In this study, we evaluated the attachment and growth behavior of these cells, using scanning- and transmission electron microscopy (SEM/TEM). Smooth and microgrooved polystyrene substrates were made (groove depth 0.5-1.5 microm, groove- and ridge width 1-10 microm). On these substrates, osteoblast-like cells were cultured for periods up to 16 days. SEM showed that the cells, and their extensions, closely followed the surface on smooth and wider grooved (>5 microm) substrates. In contrast, narrow grooves (<2 microm) were bridged. After 16 days of incubation, the matrix showed extensive deposition of collagen fibrils, and the formation of calcified nodules. With TEM it was shown that on the smooth and wider grooved substrates, focal adhesions were spread throughout the surface. However, on narrow grooves focal adhesions were always positioned on the edges of surface ridges only. Apparently, most extracellular matrix (ECM) was produced by the cells that directly adhered to the substrate. Deposition of ECM was seen in the surface grooves, as well as in between the cell layers. On basis of the current study and previous experiments, we conclude that microgrooves are able to influence bone cell behavior by (1) determining the alignment of cells and cellular extensions, (2) altering the formation and placement of cell focal adhesions, and (3) altering ECM production. Therefore, microgrooved surfaces seem interesting to be applied on bone-anchored implants.     

Contact guidance of rat fibroblasts on various implant materials.

Providing a substrate surface with micrometer-sized parallel grooves influences the behavior of cells growing on such substrates in vitro. Cells elongate in the direction of the groove and migrate guided by the grooves. It has been suggested that cellular alignment on microgrooves is predominantly dependent on groove dimensions and that surface chemical variation of the substrate material has little effect. Therefore we seeded primary rat dermal fibroblasts (RDF) on smooth and microgrooved (groove width 1-10 microm, depth 0.5 microm) polystyrene (PS), poly-L-lactic acid (PLA), silicone (SIL), and titanium (Ti) substrates. The production process was found to be more accurate for PS and PLA than for SIL and Ti substrates. A proliferation study, scanning electron microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed differences between RDF behavior on the materials. Our conclusions are (1) the accuracy of microtexture production by casting depends greatly on the material used; (2) even if no sharp discontinuities are present, microtextures still are potent tools for inducing contact guidance; and (3) besides surface texture, surface chemistry has a definitive influence on cell morphology.     

Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering

The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition.We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS.In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal organization than on PLA and TCPS. We propose that this difference in ECM composition is functionally related to the enhanced cell adhesion observed on PLAGA. There is initial evidence that specific composition of the PLAGA polymer favors the ECM. Future studies will seek to optimize ECM production on these matrices for bone tissue engineering applications.     

The ultrastructure of the bone-hydroxyapatite interface in vitro.

Rat bone marrow cells were cultured on plasma-sprayed hydroxyapatite (HA). The cells formed a mineralized extracellular matrix (ECM) that exhibited several characteristics of bone tissue. The interface between this mineralized ECM and the HA was studied at the ultrastructural level with scanning and transmission electron microscopy and x-ray microanalysis. Initially, the deposition of a globular, afibrillar matrix was observed on HA. This was followed by the integration of collagen fibers in this matrix and their subsequent mineralization. At the bone-HA interface two distinctly different interfacial structures were observed. An electron-dense layer with a thickness of 20-60 nm was regularly present, which contained both organic and inorganic material and was rich in glycosaminoglycans. The interfaces differed however, in the presence or absence of an amorphous zone which was free of collagen fibers and had an average thickness of 0.7-0.8 microns. It was frequently seen interposed between the electron-dense layer and the hydroxyapatite. Similar interfacial structures have also been described in the in vivo environment, where they were referred to as lamina limitans-like or cement linelike. From the results of this study, it can be concluded that the described in vitro system is a suitable model to study bone-biomaterial interactions.     

Biodegradable microgrooved polymeric surfaces obtained by photolithography for skeletal muscle cell orientation and myotube development

During tissue formation, skeletal muscle precursor cells fuse together to form multinucleated myotubes. To understand this mechanism, in vitro systems promoting cell alignment need to be developed; for this purpose, micrometer-scale features obtained on substrate surfaces by photolithography can be used to control and affect cell behaviour. This work was aimed at investigating how differently microgrooved polymeric surfaces can affect myoblast alignment, fusion and myotube formation in vitro. Microgrooved polymeric films were obtained by solvent casting of a biodegradable poly-l-lactide/trimethylene carbonate copolymer (PLLA–TMC) onto microgrooved silicon wafers with different groove widths (5, 10, 25, 50, 100 μm) and depths (0.5, 1, 2.5, 5 μm), obtained by a standard photolithographic technique. The surface topography of wafers and films was evaluated by scanning electron microscopy. Cell assays were performed using C2C12 cells and myotube formation was analysed by immunofluorescence assays. Cell alignment and circularity were also evaluated using ImageJ software. The obtained results confirm the ability of microgrooved surfaces to influence myotube formation and alignment; in addition, they represent a novel further improvement to the comprehension of best features to be used. The most encouraging results were observed in the case of microstructured PLLA–TMC films with grooves of 2.5 and 1 μm depth, presenting, in particular, a groove width of 50 and 25 μm.     

Effects of multigrooved surfaces on fibroblast behavior

Microgrooves have been investigated as substrates for the control of cell alignment. However, they are relatively too narrow and shallow for controlling the orientation of extracellular matrices (ECM) such as collagen. Multigrooves, a combination of microgrooves and macrogrooves, are expected to be able to control the orientation of both cells and ECM. This study investigated a method for fabricating multigrooves and evaluated fibroblast behavior on these novel surfaces. Multigrooved patterns were fabricated on a gold-alloy metal die, in which 90-degree V-shaped microgrooves with a 2-microm pitch were cut on trapezoidal macrogrooves. The macrogrooves had a 50- microm ridge width, a 50-microm wall width, a 50-microm bottom width, and a 25-microm depth. The grooves were made by an ultraprecision micromachine using a single crystal diamond. This metal die served as a template for making surface replicas from polystyrene. Microgrooved and smooth polystyrene replicas also were prepared as comparative substrates. Mouse fibroblast L929 cells were cultured in each type of replica substrate for 7 to 21 days. After these periods, the cells were fixed with 2.5% glutaraldehyde, treated with conventional methods, and, finally, observed by SEM. Confocal laser scanning microscopy was performed to investigate ECM formation. The multigrooved metal die exhibited the desired sharp configuration without defects. The dimensional values of the multigrooves on the polystyrene replicas were almost the same as the designed values. The fibroblasts on the multigrooved and microgrooved substrates were aligned parallel to the surface grooves after 7 days of incubation. In contrast to the microgrooved and flat surfaces, a dense extracellular matrix was produced along the multigrooves after 21 days of incubation. These results suggest that multigrooves can control the orientation of ECM as well as cells and thus enhance the production of ECM.     

The effect of combined cyclic mechanical stretching and microgrooved surface topography on the behavior of fibroblasts

Under the influence of mechanical stress, cultured fibroblasts have a tendency to orient themselves perpendicular to the stress direction. Similar cell alignment can be induced by guiding cells along topographical clues, like microgrooves. The aim of this study was to evaluate cell behavior on microgrooved substrates, exposed to cyclic stretching. We hypothesized that cellular shape is mainly determined by topographical clues. On basis of earlier studies, a 10-microm wide square groove, and a 40-microm wide V-shaped groove pattern were used. Smooth substrates served as controls. Onto all substrates fibroblasts were cultured and 1-Hz cyclic stretching was applied (0, 4, or 8%) for 3-24 h. Cells were prepared for scanning electron microscopy, immunostaining of filamentous actin, alignment measurements, and PCR (collagen-I, fibronectin, alpha1- and beta1-integrins). Results showed that cells aligned on all grooved surfaces, and fluorescence microscopy showed similar orientation of intracellular actin filaments. After 3 h of stretch, cellular orientation started to commence, and after 24 h the cells had aligned themselves almost entirely. Image analysis showed better orientation with increasing groove depth. Statistical testing proved that the parameters groove type, groove orientation, and time all were significant, but the variation of stretch force was not. Substrates with microgrooves perpendicular to the stretch direction elicit a better cell alignment. The expression of beta1-integrin and collagen-I was higher in the stretched samples. In conclusion, we can maintain our hypothesis, as microgrooved topography was most effective in applying strains relative to the long axis of the cell, and only secondary effects of stretch force were present.     

Connective-tissue responses to defined biomaterial surfaces. I. Growth of rat fibroblast and bone marrow cell colonies on microgrooved substrates

Surface microgeometry plays a role in tissue-implant surface interactions, but our understanding of its effects is incomplete. Substrate microgrooves strongly influence cells in vitro, as evidenced by contact guidance and cell alignment. We studied "dot" colonies of primary fibroblasts and bone marrow cells that were grown on titanium-coated, microgrooved polystyrene surfaces that we designed and produced. Rat tendon fibroblast and rat bone marrow colony growth and migration varied (p < 0.01) by microgroove dimension and slightly by cell type. We observed profoundly altered morphologies, reduced growth rates, and directional growth in colonies grown on microgrooved substrates, when compared with colonies grown on flat, control surfaces (p < 0.01). The cells in our colonies grown on microgrooved surfaces were well aligned and elongated in the direction parallel to the grooves and colonies. Our "dot" colony is an easily reproduced, easily measured and artificial explant model of tissue-implant interactions that better approximates in vivo implant responses than culturing isolated cells on biomaterials. Our results correlate well with in vivo studies of titanium dioxide-coated polystyrene, titanium, and titanium alloy implants with controlled microgeometries. Microgrooves and other surface features appear to directionally or spatially organize cells and matrix molecules in ways that contribute to improved stabilization and osseointegration of implants.     

Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration

3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.     

Alignment of osteoblast-like cells and cell-produced collagen matrix induced by nanogrooves

Alignment of bone cells and collagen matrix is closely related to the anisotropic mechanical properties of bone. Intact scaffolds that promote osteoblast differentiation and mineralization in the preferred direction offer promise in the generation of biomimetic bone tissue. In this study, we examined the alignment of osteoblast-like cells and collagen fibers guided by nanogrooves. Nanoscale groove-ridge patterns (approximately 300 nm in periodicity, 60-70 nm in depth) on the surface of polystyrene (PS) were made by polarized Nd:YAG laser irradiation, at a wavelength of 266 nm. The influence of such "nanoscale features" on the orientation and alignment of cells and their mineralized collagen matrix was investigated, using rabbit mesenchymal stem cell (MSC)-derived osteoblast-like cells. The cells and actin stress fibers were aligned and elongated along the direction of the nanogrooves. In addition, the alignment of collagen matrix was also influenced by underlying nanogrooves. The results suggested that nanoscale fibrous cues in the longitudinal direction might contribute to the aligned formation of bone tissue. This may provide an effective approach for constructing biomimetic bone tissue.     

Venous shear stress enhances platelet mediated staphylococcal adhesion to artificial and damaged biological surfaces

We investigated the role of blood components in the adhesion of staphylococci to biological and artificial surfaces under well-defined flow conditions by using the Cone and Plate(let) Analyzer. An enzyme-linked immunosorbent assay-like binding assay with biotinylated bacteria determined the extent of bacterial adhesion to subendothelial extracellular matrix (ECM), polystyrene (PS) and adult bovine aortic endothelial (ABAE) cell monolayer. The presence of adsorbed plasma proteins on PS and ECM did not increase and in some cases reduced staphylococcal adhesion under flow conditions (200s(-1)). However, their presence on ABAE cells increased bacterial adhesion but to a level still lower than the adhesion to PS and ECM. In contrast, adhered platelets significantly increased staphylococcal adhesion to both PS and ECM, but did not affect the adhesion to ABAE cells. Furthermore, bacterial adhesion to the platelets coated ECM and PS under flow conditions (200s(-1)) was increased by 1.4 to 2.6-fold compare to static conditions. The platelet-enhanced bacterial adhesion was markedly inhibited by blockade of the platelet GPIb receptor. In conclusion, staphylococcal extensive adhesion to ECM and PS surfaces is increased by venous flow and mediated by surface adhered activated platelets via a GPIb dependent mechanism. On the other hand, ABAE cells demonstrated limited bacterial adhesion that is mediated by adsorbed plasma proteins. Our results suggest that under physiological venous flow conditions the intact vessel wall is less prone for bacterial adhesion than damaged vessel wall.     

Modulation of osteogenic properties of biodegradable polymer/extracellular matrix scaffolds generated with a flow perfusion bioreactor

In this study, composite scaffolds consisting of both synthetic and natural components with controllable properties were generated by incorporating mineralized extracellular matrix (ECM) and electrospun poly(ε-caprolactone) (PCL) microfiber scaffolds. Mesenchymal stem cells (MSCs) were cultured on PCL scaffolds under flow perfusion conditions with culture medium supplemented with dexamethasone to investigate the effect of culture duration on mineralized extracellular matrix deposition. MSCs differentiated down the osteogenic lineage and produced extracellular matrix with different compositions of mineral, collagen, and glycosaminoglycan with distinct morphologies at various stages of osteogenesis. To determine whether the presence and maturity of mineralized extracellular matrix influences osteogenic differentiation in vitro, PCL/ECM constructs were decellularized to yield PCL/ECM composite scaffolds that were subsequently seeded with MSCs and cultured in the absence of dexamethasone. The presence of mineralized matrix reduced cellular proliferation while stimulating alkaline phosphatase activity with increasing amounts of calcium deposition over time. PCL/ECM composite scaffolds containing the most mature mineralized matrix resulted in the most rapid increase and highest levels of alkaline phosphatase activity and calcium deposition compared to all other scaffold groups. Therefore, we demonstrate that mineralized extracellular matrix generated under controlled flow perfusion conditions can impart osteogenic properties to an osteoconductive polymer scaffold, and that the maturity of this matrix influences osteogenic differentiation in vitro, even in the absence of dexamethasone.     

Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix

Materials based on synthetic polymers can be extensively tailored in their physical properties but often suffer from limited biological functionality. Here we tested the hypothesis that the biological performance of 3D synthetic polymer-based scaffolds can be enhanced by extracellular matrix (ECM) deposited by cells in vitro and subsequently decellularized. The hypothesis was tested in the context of bone graft substitutes, using polyesterurethane (PEU) foams and mineralized ECM laid by human mesenchymal stromal cells (hMSC). A perfusion-based bioreactor system was critically employed to uniformly seed and culture hMSC in the scaffolds and to efficiently decellularize (94% DNA reduction) the resulting ECM while preserving its main organic and inorganic components. As compared to plain PEU, the decellularized ECM-polymer hybrids supported the osteoblastic differentiation of newly seeded hMSC by up-regulating the mRNA expression of typical osteoblastic genes (6-fold higher bone sialoprotein; 4-fold higher osteocalcin and osteopontin) and increasing calcium deposition (6-fold higher), approaching the performance of ceramic-based materials. After ectopic implantation in nude mice, the decellularized hybrids induced the formation of a mineralized matrix positively immunostained for bone sialoprotein and resembling an immature osteoid tissue. Our findings consolidate the perspective of bioreactor-based production of ECM-decorated polymeric scaffolds as off-the-shelf materials combining tunable physical properties with the physiological presentation of instructive biological signals.     

Student Award for Outstanding Research Winner in the Ph.D. Category for the 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: The interplay of bone-like extracellular matrix and TNF-α signaling on in vitro osteogenic differentiation of mesenchymal stem cells

As an initial step in the development of a bone tissue engineering strategy to rationally control inflammation, we investigated the interplay of bone-like extracellular matrix (ECM) and varying doses of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) on osteogenically differentiating mesenchymal stem cells (MSCs) cultured in vitro on 3D poly(ε-caprolactone) (PCL) microfiber scaffolds containing pregenerated bone-like ECM. To generate the ECM, PCL scaffolds were seeded with MSCs and cultured in medium containing the typically required osteogenic supplement dexamethasone. However, since dexamethasone antagonizes TNF-α, the interplay of ECM and TNF-α was investigated by culturing na?ve MSCs on the decellularized scaffolds in the absence of dexamethasone. MSCs cultured on ECM-coated scaffolds continued to deposit mineralized matrix, a late stage marker of osteogenic differentiation. Mineralized matrix deposition was not adversely affected by exposure to TNF-α for 4-8 days, but was significantly reduced after continuous exposure to TNF-α over 16 days, which simulates the in vivo response, where brief TNF-α signaling stimulates bone regeneration, while prolonged exposure has damaging effects. This underscores the exciting potential of PCL/ECM constructs as a more clinically realistic in vitro culture model to facilitate the design of new bone tissue engineering strategies that rationally control inflammation to promote regeneration.     

Initial cell pre-cultivation can maximize ECM mineralization by human mesenchymal stem cells on silk fibroin scaffolds

Fast remineralization of bone defects by means of tissue engineering is one of many targets in orthopedic regeneration. This study investigated the influence of a range of pre-culture durations for human bone marrow derived mesenchymal stem cells (hMSC) before inducing differentiation into osteoblast-like cells. The aim was to find the conditions that lead to maximal extracellular matrix (ECM) mineralization, in terms of both amount and best distribution. Additionally, the influence of silk fibroin scaffold pore size on mineralization was assessed. The formation of mineralized ECM by hMSCs cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified for up to 72 days in culture using non-invasive time-lapse micro-computed tomography (micro-CT). ECM mineralization increased linearly 3 weeks after the beginning of the experiment with addition of differentiation medium. Biochemical end-point assays measured the amount of DNA, calcium deposits, alkaline phosphatase activity and cell metabolic activity to corroborate the hypothesis that an initial pre-culture period of hMSCs on silk fibroin scaffolds can accelerate mineralized ECM formation. According to the micro-CT analysis mineralization on silk fibroin scaffolds with pores of 112-224 μm diameter was most efficient with an initial cell pre-culture period of 9 days, showing 6.87±0.81× higher mineralization values during the whole cultivation period than without an initial cell pre-culture period.     

Microfabricated grooves recapitulate neonatal myocyte connexin43 and N-cadherin expression and localization

Our objective is to alter the surface topography on which cardiac myocytes are grown in culture so that they more closely resemble their in vivo counterparts. Microtextured silicone substrata were made using photolithography and microfabrication techniques and then coated with laminin. Primary cardiac myocytes from newborn rats were plated on microgrooved and nontextured substrata. Myocytes were highly oriented on 5 microm grooves (69.8 +/- 2.0%) and significantly different, p < 0.0001, compared with randomly oriented cells grown on nontextured surfaces (2.9 +/- 0.95%; n = 19). Cells on shallower, 2 microm, grooves were slightly less well oriented (46.9 +/- 4.3%, n = 5, p < 0.001). The lateral spacings of the grooves were altered to examine changes in cell-to-cell contact by confocal immunocytochemistry and quantitative protein analysis. Connexin43 and N-cadherin were distributed around the perimeter of the myocytes plated on 10 x 5 x 5 microgrooved surfaces, similar to the localization found in the neonate. Connexin43 expression in cultures on 5 microm deep grooved substrata was equal to the neonatal heart, whereas it differed in nontextured surfaces. We conclude that it is necessary to combine groove depth (5 microm) and lateral ridge dimensions between grooves (5 microm) in order to recapitulate connexin43 and N-cadherin expression levels and subcellular localization to that of the neonate.     

Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells

The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity (fibronectin > collagen I >/= collagen IV >/= vitronectin > laminin-1) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells.