Human mesenchymal stem cell responses to hydrostatic pressure and shear stress.

The effects of mechanical stimuli to which cells are exposed in vivo are, at best, incompletely understood; in this respect, gene-level information regarding cell functions which are pertinent to new tissue formation is of special interest and importance in applications such as tissue engineering and tissue regeneration. Motivated by this need, the present study investigated the early responses of human mesenchymal stem cells (hMSCs) to intermittent shear stress (ISS) and to cyclic hydrostatic pressure (CHP) simulating some aspects of the biological milieu in which these cells exist in vivo. Production of nitric oxide (NO) and mRNA expression of several known mechanosensitive genes as well as ERK1/2 activation in the hMSC response to the two mechanical stimuli tested were monitored and compared. NO production depended on the type of the mechanical stimulus to which the hMSCs were exposed and was significantly higher after exposure to ISS than to CHP. At the conditions of NO peak release (i.e., at 0.7 Pa for ISS and 50,000 Pa for CHP), ISS was more effective than CHP in up-regulating mechanosensitive genes. ERK1/2 was activated by ISS but not by CHP. The present study is the first to report that PGTS2, IER3, EGR1, IGF1, IGFBP1, ITGB1, VEGFA and FGF2 are involved in the response of hMSCs to ISS. These findings establish that, of the two mechanical stimuli tested, ISS is more effective than CHP in triggering expression of genes from hMSCs which are bioactive and pertinent to several cell functions (such as cell differentiation and release of specific growth factors and cytokines) and also to tissue-related processes such as wound healing.

Pulmonary hemodynamic effects of antisheep serum-induced leukopenia.

Antisheep antileukocyte serum (ALS) was produced in rabbits, purified, and adsorbed against erythrocytes and platelets. The ALS was infused intra-arterially over a 3-hour period into anesthetized sheep (n = 6) prepared with lung lymph fistulas. Pulmonary vascular resistance (PVR) and pulmonary lymph flow (Qlym) increased two- to threefold while the lymph-to-plasma protein concentration ratio (L/P) did not change from baseline, suggesting an increase in pulmonary vascular permeability to proteins. The arterial leukocyte count decreased from 4,935 +/- 840 to 1,385 +/- 325 cells/microliters, the neutrophil count decreased from 1,045 +/- 265 to 340 +/- 130 cells/microliters, and the platelet count decreased from 2.8 X 10(5) +/- 0.2 X 10(5) to 0.65 X 10(5) +/- 0.12 X 10(5) cells/microliters. ALS induced neutrophil aggregation in vitro, but not platelet aggregation. In the present study, we also examined the effects of ALS-induced leukopenia on the increase in pulmonary vascular permeability after intravenous alpha-thrombin challenge to assess the role of leukocytes in mediating the increased permeability response. Sheep (n = 5) were made leukopenic by an intramuscular injection of ALS; Qlym was in the normal range 4 to 5 hours after the ALS administration. In the leukopenic group, thrombin challenge caused an increase in Qlym from 7.8 +/- 0.7 to 12.9 +/- 1.9 ml/hr (a 64% increase) while L/P decreased from 0.86 +/- 0.04 to 0.70 +/- 0.05 (a 19% decrease). In contrast, thrombin-induced intravascular coagulation in control sheep (n = 5) produced a 250% increase in Qlym with an increase in L/P. The results indicate that leukocytes are required for the increase in lung vascular permeability after thrombin-induced intravascular coagulation.

RGDS tetrapeptide binds to osteoblasts and inhibits fibronectin-mediated adhesion.

The mechanisms of osteoblast attachment to surfaces were probed using the adhesive tetrapeptide RGDS (Arg-Gly-Asp-Ser) and the related but non-adhesive RGES (Arg-Gly-Glu-Ser). Specifically, RGDS and RGES were investigated for their ability both to bind to a suspension of well-characterized neonatal rat calvarial osteoblasts and to inhibit cell attachment to fibronectin-coated microtiter plates. RGDS bound to the cells with an average Kd approximately 9.4 x 10(-4) M, and RGES bound with an average Kd approximately 3.0 x 10(-4) M; at saturation, the osteoblasts bound almost twice as much RGDS as RGES. RGDS partially inhibited cell adhesion (55% to 60%) in a competitive, dose-dependent manner. In contrast, RGES had minimal effect on cell attachment. Since complete inhibition of attachment was not observed, it is likely that a synergistic adhesion site in the fibronectin molecule and/or cell surface molecules such as proteoglycans are active in mediating osteoblast/substrate adhesion.

Inhibition of human scleral fibroblast proliferation with heparin.

Proliferation of fibroblasts is a serious problem in ocular trauma and surgical wound healing. Depending on the location of the injury, the growth of fibroblasts can lead to different problems. In glaucoma filtering surgery, fibroblast proliferation may contribute to scar tissue formation and premature wound closure. Fibroblastic growth in proliferative vitreoretinopathy may lead to the formation of preretinal membranes, which can contract, causing retinal detachment. In an effort to find a more effective method of inhibiting ocular fibroblast proliferation, we have investigated the effect of heparin, a sulfated polysaccharide, on the proliferation of fibroblasts obtained from the sclera of donor eyes. Heparin inhibits the incorporation of 3H-thymidine in a dose-dependent manner in the presence of fetal bovine serum (FBS). This inhibition is partially reversed by endothelial cell growth factor (ECGF). The heparin antagonist protamine sulfate causes a reversal of heparin inhibition and, in some instances, a significant increase in 3H-thymidine incorporation compared to serum controls. Heparin was equally effective in inhibiting cell proliferation in control and heparin-protamine sulfate-pretreated medium. These results were apparently unrelated to a direct toxic effect on cells, as a Trypan Blue exclusion assay showed no significant difference in viability when heparin treated cells were compared to control cells. Direct cell counts showed that heparin was effective in inhibiting cell proliferation over a long time period, but only if it was reinstilled every 2 days. Heparin treatment shows promise as a method for controlling fibroblast proliferation in the eye.

Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts.

Microcontact printing techniques were used to pattern circles (diameters 10. 50, 100, and 200 microm) of N1[3-(trimethoxysilyl)-propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS) borders on borosilicate glass, a model substrate. The DETA regions were further modified by immobilization of either the cell-adhesive peptides Arginine-Glycine-Aspartic Acid-Serine (RGDS) and Lysine-Arginine-Serine-Arginine (KRSR) or the non-adhesive peptides Arginine-Aspartic Acid-Glycine-Serine (RDGS) and Lysine-Serine-Serine-Arginine (KSSR). After four hours under standard cell culture conditions but in the absence of serum, adhesion of either osteoblasts or fibroblasts on surfaces patterned with the non-adhesive peptides RDGS and KSSR was random and low. In contrast, both osteoblasts and fibroblasts adhered and formed clusters onto circles modified with the adhesive peptide RGDS, whereas only osteoblasts adhered and formed clusters onto the circles modified with KRSR, a peptide that selectively promotes adhesion of osteoblasts. These results provide evidence that patterning of select peptides can direct adhesion of specific cell lines exclusively to predetermined regions on material surfaces.

Analysis of osteoblast mineral deposits on orthopaedic/dental implant metals.

Neonatal rat calvarial osteoblasts were cultured on Ti-6Al-4V, Co-Cr-Mo alloy, 316L stainless steel and polystyrene (reference substrate) in the presence of ascorbic acid and 10 mM beta-glycerophosphate for 16, 17, 18, 19, 20, 21, 24 and 28 d. Scanning electron microscopy examination revealed that osteoblasts cultured on these orthopaedic/dental implant metals synthesized and deposited an extracellular matrix containing collagenous and non-collagenous components, as well as mineral nodules of various morphologies. Energy dispersive spectrometry revealed that the mineral deposits consisted of three distinct chemical compositions: calcium phosphate, calcium-sulphur-phosphorus, and calcium only. Backscattered electron imaging demonstrated that both the calcium phosphate and calcium-only deposits were electron dense, while the calcium-sulphur-phosphorus deposits were electron translucent. X-ray diffraction analysis indicated that the bulk of the osteoblast mineral deposits was amorphous hydroxyapatite; in addition, electron diffraction analysis revealed small regions of crystalline hydroxyapatite.

Osteoblast population migration characteristics on substrates modified with immobilized adhesive peptides.

The process of cell migration is inextricably linked with the process of cell adhesion and, therefore, with cell/substrate adhesiveness. The present study adapted an under-agarose cell migration assay to quantitatively examine population migration characteristics of osteoblasts, on substrates modified with adhesive peptides, in the absence and presence of growth factors. Short-term, that is, 48 h osteoblast migration distances on substrates modified with adhesive Arg-Gly-Asp-Ser peptides were significantly (P < 0.05) less than migration distances on substrates modified with non-adhesive Arg-Asp-Gly-Ser peptides, demonstrating that osteoblast population haptokinesis was significantly decreased on substrates modified with adhesive peptides. Random motility coefficients calculated in the present study for osteoblast populations were an order of magnitude lower than a published random motility coefficient for leukocytes, proving quantitatively that, compared to leukocytes, osteoblasts migrate via haptokinesis more slowly. The 48 and 72 h osteoblast population migration differentials in the presence of an initial mass of 60 ng of basic Fibroblast Growth Factor, on substrates modified with Arg-Gly-Asp-Ser or with Arg-Asp-Gly-Ser, were larger than all other chemotactic differentials on these substrates. Quantitative investigations (such as the present study) of cell population migration characteristics on model biomaterial surfaces will become increasingly necessary as the discipline of cell/tissue engineering matures.

Osteoblast adhesion on nanophase ceramics.

Osteoblast adhesion on nanophase alumina (Al2O3) and titania (TiO2) was investigated in vitro. Osteoblast adhesion to nanophase alumina and titania in the absence of serum from Dulbecco's modified Eagle medium (DMEM) was significantly (P < 0.01) less than osteoblast adhesion to alumina and titania in the presence of serum. In the presence of 10% fetal bovine serum in DMEM osteoblast adhesion on nanophase alumina (23 nm grain size) and titania (32 nm grain size) was significantly (P < 0.05) greater than on conventional alumina (177 nm grain size) and titania (2.12 microm grain size), respectively, after 1, 2, and 4 h. Further investigation of the dependence of osteoblast adhesion on alumina and titania grain size indicated the presence of a critical grain size for osteoblast adhesion between 49 and 67 nm for alumina and 32 and 56 nm for titania. The present study provides evidence of the ability of nanophase alumina and titania to simulate material characteristics (such as surface grain size) of physiological bone that enhance protein interactions (such as adsorption, configuration, bioactivity, etc.) and subsequent osteoblast adhesion.

Examination of osteoblast-orthopaedic biomaterial interactions using molecular techniques.

Molecular techniques can be used to elucidate the effects of extended periods of cell-biomaterial interactions on the time-course and level of expression of particular genes which determine cellular phenotype. We used the polymerase chain reaction to demonstrate the expression of genes for the bone-related proteins osteocalcin, osteonectin and osteopontin by neonatal rat calvarial osteoblasts. In addition, Northern blotting was subsequently used to show that messenger RNAs encoding osteonectin and osteopontin were consistently expressed during a 5 wk period of interaction of osteoblasts with Ti-6Al-4V, a commercial brand of hydroxyapatite, and tissue culture polystyrene.

Osteoblasts on hydroxyapatite, alumina and bone surfaces in vitro: morphology during the first 2 h of attachment.

The morphological responses of individual osteoblasts as they attached and spread on hydroxyapatite, bovine bone, alumina with rough and polished surfaces, and tissue culture polystyrene in vitro were examined with scanning electron microscopy. Depending on the surface tested two different morphological sequences were observed during 2 h of adhesion. On alumina, both rough and smooth, bone, and tissue culture polystyrene the cells were round after 0.5 h, and spread radially during the next 1.5 h until they were almost flat, with a nuclear bulge. On hydroxyapatite, however, the cells were flat and circular at 0.5 h, and the edge of the cytoplasm was hardly discernable. This morphology did not change much during the subsequent 1.5 h. The observed cellular morphological response may be related to the bioreactivity of hydroxyapatite.

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.

Axonal outgrowth of hippocampal neurons on micro-scale networks of polylysine-conjugated laminin.

Microcontact printing was used to define an interconnected lattice network of polylysine-conjugated laminin, a protein-polypeptide ligate that is an effective promoter of neuron outgrowth on material surfaces. In the presence of serum proteins, rat hippocampal neurons selectively adhered to features of polylysine-conjugated laminin as narrow as 2.6 microm in width. Adhering neurons extended long axonal processes, which precisely followed and did not deviate from the prescribed patterns, demonstrating that neurons respond to this protein with high selectivity and that these techniques effectively provide long-range guidance of axonal outgrowth. Further examination of neuron response under serum-free cell culture conditions demonstrated that the outgrowth-promoting activity of polylysine-conjugated laminin was attributed to biologically active laminin. Together, these results demonstrate that polylysine-conjugated laminin provides for high-precision guidance of neuron attachment and axon outgrowth on material surfaces in a serum-independent manner. This ability to guide hippocampal neuron response in low-density, serum-free culture with high precision is valuable for the development of advanced, neuron-based devices.

Surfaces modified with covalently-immobilized adhesive peptides affect fibroblast population motility.

Cell population motility and adhesion of rat skin fibroblasts were evaluated on aminophase glass modified with covalently-immobilized biologically active peptides, specifically, either arginine glycine-aspartic acid-serine (RGDS) or tyrosine-isoleucine-glycine-serine-arginine-glycine (YIGSRG). Fibroblast population motility was decreased and adhesion was increased on substrates modified with covalently immobilized RGDS peptide compared to substrates with the covalently immobilized non-adhesive peptides arginine-glycine-glutamic acid-serine and arginine-aspartic acid-glycine-serine. Fibroblast motility was not significantly changed on substrates modified with covalently-immobilized YIGSRG peptide; however, fibroblast adhesion was decreased on that substrate.

Conditions which promote mineralization at the bone-implant interface: a model in vitro study.

This in vitro study was an investigation of osteoblast functions on glass substrates modified with the bioactive peptide Arg-Gly-Asp-Ser (RGDS) in the absence and presence of recombinant human Osteogenic Protein-1 (OP-1); control substrates were plain glass, glass modified with amine groups, and glass modified with the non-adhesive peptide Arg-Asp-Gly-Ser. In serum-free cell culture medium, osteoblasts adhered in greater numbers (P < 0.1) to glass modified with RGDS, compared to adhesion on all other substrate types tested in the present study. In the presence of serum proteins, osteoblasts adhered similarly to all substrate types examined, in the absence or presence of 100 ng ml-1 OP-1. The presence of 100 ng ml-1 OP-1 inhibited (P < 0.1) 72 h proliferation of sparsely seeded (2500 cells cm-2) cultures on all substrates examined in the present study. OP-1 (100 ng ml-1) promoted 21 day mineralization on all substrates examined; in addition, mineralization was further enhanced in osteoblast cultures grown on glass modified with the adhesive peptide RGDS. The present study establishes conditions which can be utilized in the design of dental/orthopaedic biomaterials which elicit timely, specific responses from surrounding bone tissue.

Correlation of astroglial cell function on micro-patterned surfaces with specific geometric parameters.

Microcontact printing techniques were used to modify silicon substrates with arrays of hexagonal features of N1[3-(trimethoxysilyl) propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS), which are hydrophilic, cell-adhesive and hydrophobic, non-adhesive organosilanes, respectively. In the presence of serum proteins, LRM55 cell adhesion and morphology on these modified surfaces were best correlated to the width of the cell-adhesive features. On surfaces modified with small (5 microm in width) cell-adhesive features, LRM55 cells elaborated only thin processes. As feature width was increased, cells on these surfaces exhibited increased cell spreading and elaborated wide processes. On surfaces modified with large (>35 microm in width) features, single cells adhered to and spread upon individual DETA features. In a similar fashion, LRM55 cell adhesion density increased with increasing feature width; this correlation could be represented by a simple, second-order relation, and was independent of all other measures of pattern geometry. The results of this study provide evidence that micro-patterning may be effective in controlling astrocyte interaction with implant materials.

Endothelial cell migration on surfaces modified with immobilized adhesive peptides.

Endothelial cell (EC) migration has been studied on aminophase surfaces with covalently bound RGDS and YIGSRG cell adhesion peptides. The fluorescent marker dansyl chloride was used to quantify the spatial distribution of the peptides on the modified surfaces. Peptides appeared to be distributed in uniformly dispersed large clusters separated by areas of lower peptide concentrations. We employed digital time-lapse video microscopy and image analysis to monitor EC migration on the modified surfaces and to reconstruct the cell trajectories. The persistent random walk model was then applied to analyze the cell displacement data and compute the mean root square speed, the persistence time, and the random motility coefficient of EC. We also calculated the time-averaged speed of cell locomotion. No differences in the speed of cell locomotion on the various substrates were noted. Immobilization of the cell adhesion peptides (RGDS and YIGSRG), however, significantly increased the persistence of cell movement and, thus, the random motility coefficient. These results suggest that immobilization of cell adhesion peptides on the surface of implantable biomaterials may lead to enhanced endothelization rates.

Enhanced functions of osteoblasts on nanophase ceramics.

Select functions of osteoblasts (bone-forming cells) on nanophase (materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) were investigated using in vitro cellular models. Compared to conventional ceramics, surface occupancy of osteoblast colonies was significantly less on all nanophase ceramics tested in the present study after 4 and 6 days of culture. Osteoblast proliferation was significantly greater on nanophase alumina, titania, and HA than on conventional formulations of the same ceramic after 3 and 5 days. More importantly, compared to conventional ceramics, synthesis of alkaline phosphatase and deposition of calcium-containing mineral was significantly greater by osteoblasts cultured on nanophase than on conventional ceramics after 21 and 28 days. The results of the present study provided the first evidence of enhanced long-term (on the order of days to weeks) functions of osteoblasts cultured on nanophase ceramics; in this manner, nanophase ceramics clearly represent a unique and promising class of orthopaedic/dental implant formulations with improved osseointegrative properties.

Enhanced osteoclast-like cell functions on nanophase ceramics.

Synthesis of tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits by osteoclast-like cells, the bone-resorbing cells, on nanophase (that is, material formulations with grain sizes less than 100nm) alumina and hydroxyapatite (HA) were investigated in the present in vitro study. Compared to conventional (that is, grain sizes larger than 100 nm) ceramics, synthesis of TRAP was significantly greater in osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 10 and 13 days, respectively. In addition, compared to conventional ceramics, formation of resorption pits was significantly greater by osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 7, 10, and 13 days, respectively. The present study, therefore, demonstrated, for the first time, enhanced osteoclast-like cell function on ceramic surfaces with nanometer-size surface topography.

Retinal pigment epithelial cell adhesion on novel micropatterned surfaces fabricated from synthetic biodegradable polymers.

Novel synthetic biodegradable polymer substrates with specific chemical micropatterns were fabricated from poly(DL-lactic-coglycolic acid) (PLGA) and diblock copolymers of poly(ethylene glycol) and poly(DL-lactic acid) (PEG/PLA). Thin films of PLGA and PEG/PLA supported and inhibited, respectively, retinal pigment epithelial (RPE) cell proliferation, with a corresponding cell density of 352,900 and 850 cells/cm2 after 7 days (from an initial seeding density of 15,000 cells/cm2). A microcontact printing technique was used to define arrays of circular (diameter of 50 microm) PLGA domains surrounded and separated by regions (width of 50 microm) of PEG/PLA. Reversed patterns composed of PEG/PLA circular domains surrounded by PLGA regions were also fabricated. Both micropatterned surfaces were shown to affect initial RPE cell attachment, limit cell spreading, and promote the characteristic cuboidal cell morphology during the 8-h period of the experiments. In contrast, RPE cells on plain PLGA (control films) were elongated and appeared fibroblast-like. The reversed patterns had continuous PLGA regions that allowed cell-cell interactions and thus higher cell adhesion. These results demonstrate the feasibility of fabricating micropatterned synthetic biodegradable polymer surfaces to control RPE cell morphology.

Retinal pigment epithelial cell function on substrates with chemically micropatterned surfaces.

Model substrates with desired chemical micropatterns were fabricated using a microcontact printing technique. The substrate surfaces contained organized arrays of circular glass domains with a diameter of either 10 or 50 microm surrounded and separated by regions modified with octadecyltrichlorosilane (OTS) self-assembled monolayers (SAMs). The effects of surface patterning on in vitro cell attachment, proliferation, morphology, and cytoskeletal organization were evaluated using a human retinal pigment epithelium (RPE) cell line. Both micropatterns affected initial RPE cell attachment, limited cell spreading, and promoted the characteristic cuboidal cell morphology throughout the culture period. In contrast, RPE cells on plain glass control were elongated and appeared fibroblast-like prior to confluence. In addition, cells seeded at 30,000 cell/cm2 on the patterned surfaces maintained a normal pattern of actin and cytokeratin expression, and formed confluent monolayers within 4 days of culture. The cell density increased about 30-fold on both micropatterns by day 7. These results show that it is feasible to control RPE cell shape and expression of differentiated phenotype using micropatterned surfaces.