Glycosaminoglycan mimetic peptide nanofibers promote mineralization by osteogenic cells

Bone tissue regeneration is accomplished by concerted regulation of protein-based extracellular matrix components, glycosaminoglycans (GAGs) and inductive growth factors. GAGs constitute a significant portion of the extracellular matrix and have a significant impact on regulating cellular behavior, either directly or through encapsulation and presentation of growth factors to the cells. In this study we utilized a supramolecular peptide nanofiber system that can emulate both the nanofibrous architecture of collagenous extracellular matrix and the major chemical composition found on GAGs. GAGs and collagen mimetic peptide nanofibers were designed and synthesized with sulfonate and carboxylate groups on the peptide scaffold. The GAG mimetic peptide nanofibers interact with bone morphogenetic protein-2 (BMP-2), which is a critical growth factor for osteogenic activity. The GAG mimicking ability of the peptide nanofibers and their interaction with BMP-2 promoted osteogenic activity and mineralization by osteoblastic cells. Alkaline phosphatase activity, Alizarin red staining and energy dispersive X-ray analysis spectroscopy indicated the efficacy of the peptide nanofibers in inducing mineralization. The multifunctional and bioactive microenvironment presented here provides osteoblastic cells with osteogenic stimuli similar to those observed in native bone tissue.     

The effect of ligand type and density on osteoblast adhesion, proliferation, and matrix mineralization

Polystyrene surfaces grafted with a nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] were modified with several peptide ligands adapted from bone sialoprotein (BSP). IPNs were modified with both single ligands and ligand blends to study the correlation between a simple metric, ligand-receptor adhesion strength, and the extent of matrix mineralization for osteoblast like cells (rat calvarial osteoblasts). The ligands studied included RGD cell-binding [CGGNGEPRGDTYRAY (l-RGD), CGGEPRGDTYRA (s2-RGD), CGPRGDTYG (lc-RGD), cyclic(CGPRGDTYG) (c-RGD), and CGGPRGDT (s-RGD)], heparin binding (CGGFHRRIKA), and collagen binding (CGGDGEAG) peptides, with the appropriate controls. Adhesion strength scaled with ligand density (1-20 pmol/cm(2)) and was dependent on ligand type with the following trend: l-RGD > s2-RGD approximately c-RGD > s-RGD approximately lc-RGD > FHRRIKA approximately DGEA. Independent of ligand density, % matrix mineralization varied with ligand type resulting in the following trend: lc-RGD > s2-RGD > l-RGD approximately c-RGD > s-RGD > FHRRIKA. The Tyr (Y) residue immediately following the RGD cell-binding domain proved to be critical for stable cell proliferation and mineralization, since removal of this residue resulted in erratic cell attachment and mineralization behavior. The minimum BSP sequence necessary for strong adhesion and extensive mineralization was CGGEPRGDTYRA; the minimal sequence suitable for extensive mineralization but lacking strong adhesion was CGPRGDTYG. The cyclic peptide (c-RGD) had much greater adhesion strength compared to its linear counterpart (lc-RGD). The calculated characteristic adhesion strength (F(70)) obtained using a centrifuge adhesion assay proved to be a poor metric for predicting % mineralized area; however, in general, surfaces possessing a F(70) > 100g promoted extensive matrix mineralization. Percent mineralization and number of mineralized nodules scaled with number of cells seeded suggesting a critical dependence on the initial number of osteoprogenitors in culture. This study demonstrates matrix mineralization dependence on ligand type, ligand density, and adhesion strength. The high-throughput character of these surfaces allowed efficient investigation of multiple ligands at multiple densities providing an excellent tool for studying ligand-receptor interactions under normal cell culture conditions with serum present.     

Cellular alignment by grafted adhesion peptide surface density gradients

The extracellular matrix and extracellular matrix-associated proteins play a major role in growth and differentiation of tissues and organs. To date, few methods have been developed that allow researchers to examine the affect of surface density gradients of adhesion molecules in a controlled manner. Fibroblasts cultured on surfaces with a surface density gradient of RGD peptide aligned parallel to the gradient while fibroblasts on constant density RGD surfaces spread but did not align as has been shown in numerous earlier studies. Not only did fibroblasts align on the gradient surfaces, but they also showed significantly greater elongation than on constant density peptide surfaces or on control surfaces. This type of method is easy to replicate and can be used by laboratories interested in investigating alignment of various cell types without mechanical force or other stimulation, and without cell-cell interaction or for investigation of affects of surface density gradients of molecules on cellular biochemistry and biophysics. This method also has potential applications for developing scaffolds for tissue engineering applications where cellular alignment is necessary.     

The effect of natural extracellular matrix deposited on synthetic polymers on cultured primary hepatocytes

It is well known that natural extracellular matrix (ECM) molecules are deposited on the surface of biomaterials during culture of cells and affect cellular behaviors. However, it has not been fully understood what kinds of ECM molecules are deposited on the surface of biomaterials although the cellular behaviors were affected by deposited ECM. In this study, to investigate the effect of deposited natural ECM on behaviors of hepatocytes cultured on biomaterials such as poly (N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-gluconamide) (PVLA) as a hepatocyte-specific matrix and poly (L-lysine) (PLL) as a non-specific one during the culture of hepatocytes in vitro, we investigated expression pattern of ECM genes and adsorption of ECM molecules onto PVLA- and PLL-coated surfaces. It was found that the expression levels of type I collagen and fibronectin genes in the hepatocytes cultured on PVLA-coated surface were different from them in the hepatocytes cultured on PLL-coated one. Also, the results showed that laminin was dominantly deposited on PVLA-coated surface whereas fibronectin was dominantly deposited on PLL-coated one. Hepatocytes maintained liver-specific functions on PVLA- and laminin-coated surfaces. It is thought that deposited laminin during the culture of hepatocytes affects the liver-specific functions of hepatocytes cultured on PVLA-coated surface.     

Effect of nicotine in matrix mineralization by human bone marrow and Saos-2 cells cultured on the surface of plasma-sprayed titanium implants

Smoking has an established negative impact in the clinical outcome of dental implants. This work analyses the response of human osteoblastic cells to nicotine, at the surface of plasma-sprayed commercial titanium implants. Human bone marrow (HBM) and Saos-2 cells, seeded on the surface of titanium implants and cultured in experimental conditions favoring osteoblastic differentiation, were exposed continuously to nicotine (0.0001 to 0.5 mg mL(-1)) and characterized for cell proliferation and function. Exposure of HBM cells resulted in increased cell proliferation, higher alkaline phosphatase (ALP) activity, and earlier onset of matrix mineralization at levels up to 0.2 mg mL(-1), an initial inhibitory effect in cell growth and functional activity followed by a recovery in the presence of 0.3 mg mL(-1) and a dose-dependent deleterious effect at higher levels. By contrast, exposure to nicotine did not affect cell proliferation of Saos-2 cells at levels up to 0.2 mg mL(-1), and caused only a small positive effect in ALP activity in the presence of 0.05 and 0.1 mg mL(-1); however, matrix mineralization by Saos-2 cells also occurred earlier in the cultures exposed to levels of nicotine up to 0.1 mg mL(-1). Higher concentrations caused dose-dependent inhibitory effects. Considering the high diffusion potential of nicotine, results suggest a local role of nicotine in modulating bone formation events at the implant surface.     

The effect of glow discharge plasma surface modification of polymers on the osteogenic differentiation of committed human mesenchymal stem cells

Little is known of the effect of material surfaces on stem cell differentiation. The present study has addressed the hypothesis that the interaction of mesenchymal stem cells (MSCs) with material surfaces modified by glow discharge plasma is a major regulator of osteogenic differentiation. We found that biaxially oriented polypropylene (BOPP) plasma treated in ammonia significantly reduced up-regulation of expression of osteogenic marker genes, such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). In contrast, ALP expression was up-regulated when cultured on treated Nylon-6 polyamide (Ny-t) but was substantially reduced when cultured on its pristine counterpart (Ny-p) on day 3. On day 7, ALP expression was down-regulated with MSCs cultured on Ny-t although its expression level was up again on day 14. BSP was expressed weakly on day 3, but was up-regulated when cultured on Ny-t and Ny-p. Its expression reached its maximum on day 14 when cultured on a polystyrene control, while it was cyclically up-regulated on Ny-t. Similarly, there was a slight increase in OC expression when MSCs were cultured on Ny-t and Ny-p on day 3, when compared to control. Thus, the nature of the surface can directly influence MSCs differentiation, ultimately affecting the quality of new tissue formation with BOPP-t suppressing osteogenic differentiation.     

The effect of surface treatment on the surface texture and contact angle of electrochemically deposited hydroxyapatite coating and on its interaction with bone-forming cells

This work demonstrates the effects of both surface preparation and surface post-treatment by exposure to electron beam on the surface texture, contact angle and the interaction with bone-forming cells of electrochemically deposited hydroxyapatite (HAp) coating. Both the surface texture and the contact angle of the ground titanium substrate changed as a result of either heat treatment following soaking in NaOH solution or soaking in H₂O₂ solution. Consequently, the shape of the current transients during potentiostatic deposition of HAp changed, and the resulting coatings exhibited different surface textures and contact angles. The developed interfacial area ratio Sdr and the core fluid retention index Sci were found more reliable than the mean roughness R_a and the root-mean-square roughness Z_rms in correlating the adhesion of the coating to the metal substrate and the cellular response with surface texture. The NaOH pretreatment provided the highest surface area and induced the highest cell attachment, even though the H₂O₂ treatment provided the highest hydrophilicity to the metal substrate. Electrodeposition at pH 6 was found preferable compared to electrodeposition at pH 4.2. The ability to modify the cellular response by exposure to unique electron-beam surface treatment was demonstrated. The very high hydrophilicity of the as-deposited HAp coating enhanced its bioactivity.     

High concentration of residual aluminum oxide on titanium surface inhibits extracellular matrix mineralization

In the present study we characterized titanium (Ti) surfaces submitted to different treatments and evaluated the response of osteoblasts derived from human alveolar bone to these surfaces. Five different surfaces were evaluated: ground (G), ground and chemical etched (G1-HF for 60 s), sand blasted (SB-Al(2)O(3) particles 65 mum), sand blasted and chemical etched (SLA1-HF for 60 s and SLA2-HF for 13 s). Surface morphology was evaluated under SEM and roughness parameters by contact scanning instrument. The presence of Al(2)O(3) was detected by EDS and the amount calculated by digital analyses. Osteoblasts were cultured on these surfaces and it was evaluated: cell adhesion, proliferation, and viability, alkaline phosphatase activity, total protein content, and matrix mineralization formation. Physical and chemical treatments produced very different surface morphologies. Al(2)O(3) residues were detected on SB and SLA2 surfaces. Only matrix mineralization formation was affected by different surface treatments, being increased on rough surface (SLA1) and reduced on surface with high amount of Al(2)O(3) residues (SB). On the basis of these findings, it is possible to conclude that high concentration of residual Al(2)O(3) negatively interfere with the process of matrix mineralization formation in contact with Ti implant surfaces.     

Comparison of in vitro mineralization by murine embryonic and adult stem cells cultured in an osteogenic medium

Nearly half a million bone-grafting procedures occurred in the United States in the year 2000. Tissue-engineered bone substitutes may mitigate difficulties associated with current grafting options. Embryonic stem cells (ESCs) could be a potential cell source for bone substitutes; however, direct comparisons between ESCs and other cell sources are lacking. Here we provide a direct, long-term, in vitro comparison of mineralization processes in adult, marrow-derived, mesenchymal stem cells (MSCs) and ESCs from the 129/Sv+c/+p mouse strain. MSCs were observed to grow at a slower rate than ESCs. MSCs expressed seven times more alkaline phosphatase (AP) per cell than did ESCs and immediately showed type I collagen and osteocalcin production. ESCs also produced type I collagen and osteocalcin, but production was delayed. Mineral deposition by ESCs was nearly 50 times higher than by MSCs. Spectroscopic analysis showed the calcium-to-phosphorus ratio (Ca:P) of the ESC mineral (1.26:1) to be significantly higher than that of the MSCs (0.29:1), but still 25% lower than hydroxyapatite (1.67:1). Addition of basic fibroblast growth factor significantly inhibited AP expression, mineral deposition, and Ca:P ratios in MSCs and had little effect on ESCs. These functional characteristics may assist with cell selection for purposes of bone tissue engineering.     

Differential regulation of osteogenic differentiation of stem cells on surface roughness gradients

Tissue engineering using scaffold-cell constructs holds the potential to develop functional strategies to regenerate bone. The interface of orthopedic implants with the host tissues is of great importance for its later performance. Thus, the optimization of the implant surface in a way that could stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) is of significant therapeutic interest. The effect of surface roughness of polycaprolactone (PCL) on the osteogenic differentiation of human bone-marrow MSCs was investigated. We prepared surface roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range (∼0.5–4.7 μm), and mean distance between peaks (RS_m) gradually varying from ∼214 μm to 33 μm. We analyzed the degree of cytoskeleton spreading, expression of alkaline phosphatase, collagen type 1 and mineralization. The response of cells to roughness divided the gradient into three groups of elicited stem cell behavior: 1) faster osteogenic commitment and strongest osteogenic expression; 2) slower osteogenic commitment but strong osteogenic expression, and 3) similar or inferior osteogenic potential in comparison to the control material. The stem-cell modulation by specific PCL roughness surfaces highlights the potential for creating effective solutions for orthopedic applications featuring a clinically relevant biodegradable material.     

The enamel protein ODAM promotes mineralization in a collagen matrix

ABSTRACT

Purpose/aim of the study: Odontogenic ameloblast-associated protein (ODAM) is predominantly expressed during the maturation stage of enamel formation and interacts strongly with amelotin (AMTN). AMTN is involved in enamel mineralization, but the effect of ODAM on mineralization has not been investigated. This study determined whether ODAM was able to induce hydroxyapatite (HA) mineralization in modified simulated body fluid (SBF) and in a collagen matrix in vitro. Materials and methods: To monitor the kinetics of calcium phosphate mineralization, recombinant human (rh) ODAM protein in SBF buffer was incubated at 37°C and a light-scattering assay was conducted at intervals. To investigate the nucleation of ODAM in collagen matrix, the ODAM-impregnated collagen hydrogel was incubated in SBF buffer for 24 hours. Bovine serum albumin (BSA) was used as negative control. Mineral deposits were visualized using electron microscopy. Results: The presence of rh-ODAM protein in SBF resulted in higher light-scattering values after 18–24 hours. Calcium phosphate precipitates were observed on the surface of the ODAM-treated, but not BSA-treated collagen hydrogel after 24 hours in SBF. TEM and SAED analyses showed that these crystals consisted of needle-like HA. Conclusion: Similar to AMTN, ODAM is able to promote HA nucleation in a dose-dependent manner in SBF, and even outside of its biological context in vitro.     

Adjuvant effect of gamma-inulin is mediated by C3 fragments deposited on antigen-presenting cells

The adjuvant effect of gamma-inulin, a strong activator of the alternative complement pathway, is well-known, but its exact mechanism is not revealed yet. Here, we show that macrophages, isolated from the peritoneal cavity of gamma-inulin-injected mice and used as antigen-presenting cells, enhance the proliferation of antigen-specific T-cells up to 2.5-fold when compared with macrophages of non-treated animals. This effect is abrogated by the presence of anti-C3 F(ab')2 fragments and by prior decomplementation of the donor animals with CVF. It is demonstrated that treatment of mice with the adjuvant results in deposition of C3-fragments onto the surface of peritoneal macrophages, as does in vitro incubation of the cells with gamma-inulin in the presence of fresh autologous serum. Prior incubation of macrophages with gamma-inulin plus serum in vitro enhances subsequent C3 production. Because it has been shown earlier that CR1/2 expressed on activated T-cells and interacting with covalently bound C3-fragments plays an important role in the augmentation of the adaptive response, our present results reveal a mechanism that contributes to the adjuvant effect of gamma-inulin and point to a further link between innate and adaptive immunity.     

补骨脂素对大鼠骨髓间充质干细胞成骨分化的影响

目的研究不同浓度的补骨脂素对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)成骨分化的影响,为临床应用补骨脂素治疗骨质疏松症提供理论依据。方法选取8周龄健康雄性SD大鼠,取双侧股骨、胫骨,对大鼠的BMMSCs进行分离、原代培养和细胞表型鉴定。取第3代大鼠BMMSCs,体外利用成骨诱导培养基进行不同浓度补骨脂素的药物诱导,MTT法检测不同浓度的补骨脂素对大鼠BMMSCs生长的影响;通过碱性磷酸酶(ALP)活性测定、Western blot法和茜素红染色方法评价不同浓度补骨脂素对大鼠BMMSCs成骨分化能力。结果第3代大鼠BMMSCs表面抗原符合干细胞鉴定标准,成骨诱导后给予15μmol/L补骨脂素对大鼠BMMSCs的增殖作用最佳,ALP活性、Runx-2表达和钙化结节数目均明显高于经典成骨诱导组、5μmol/L和10μmol/L和20μmol/L补骨脂素组。结论15μmol/L补骨脂素成骨诱导促进BMMSCs向成骨分化,从而起到防治骨质疏松的作用。     

Effect of nano-structured bioceramic surface on osteogenic differentiation of adipose derived stem cells

Tissue engineering strategies to construct vascularized bone grafts potentially revolutionize the treatment of massive bone loss. The surface topography of the grafts plays critical roles on bone regeneration, while adipose derived stem cells (ASCs) are known for their capability to promote osteogenesis and angiogenesis when applied to bone defects. In the present study, the effects of hydroxyapatite (HAp) bioceramic scaffolds with nanosheet, nanorod, and micro-nano-hybrid (the hybrid of nanorod and microrod) surface topographies on attachment, proliferation and osteogenic differentiation, as well as the expression of angiogenic factors of rat ASCs were systematically investigated. The results showed that the HAp bioceramic scaffolds with the micro-/nano-topography surfaces significantly enhanced cell attachment and viability, alkaline phosphatase (ALP) activity, and mRNA expression levels of osteogenic markers and angiogenic factors of ASCs. More importantly, the biomimetic feature of the hierarchical micro-nano-hybrid surface topography showed the highest stimulatory effect. The activation in Akt signaling pathway was observed in ASCs cultured on HAp bioceramics with nanorod, and micro-nano-hybrid surface topographies. Moreover, these induction effects could be repressed by Akt signaling pathway inhibitor LY294002. Finally, the in vivo bone regeneration results of rat critical-sized calvarial defect models confirmed that the combination of the micro-nano-hybrid surface and ASCs could significantly enhance both osteogenesis and angiogenesis as compared with the control HAp bioceramic scaffold with traditional smooth surface. Our results suggest that HAp bioceramic scaffolds with micro-nano-hybrid surface can act as cell carrier for ASCs, and consequently combine with ASCs to construct vascularized tissue-engineered bone.     

Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells.

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.     

The immune response in stationary suspension cultures containing different numbers of cells. The surface density effect.

Correlation between the number of dissociated spleen cells, incubated with antigen in a modified Mishell and Dutton system, and the number of antibody-forming cells (AFC) produced as a result of incubation has been studies in mice of C57Bl/6 strain. It has been shown that when suspension densities are increased 2- to 4-fold, the number of AFC formed is very often reduced 10- to 100-fold, although the percentage of viable cells recovered at the end of incubation is not significantly diminished. The observed reduction of AFC formation in cultures containing increased numbers of cells, designated here the surface density effect, was found to be expressed more by spleen cells of unimmunized than of immunized mice. Inhibition was dependent on the thickness of the cell layer formed on the bottom of the incubation vessel (cells per square centimeter), rather than on the cell: volume ratio of cultures. The effect was not due to a deficit of antigen or nutrition and could not be reproduced by adding of incubation media conditioned by dense cultures. It was determined not by impairment of clone induction but by inhibition of subsequent proliferation. This suppression is reversible and can be reversed by decreasing the cell density, even after 3 days of culture.     

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: The effect of silanol groups

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si–OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si–OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL–Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL–Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL–Si scaffolds occur for up to 2 weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL–Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies.     

Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell’s capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.