Heparin Enriched-WPI Coating on Ti6Al4V Increases Hydrophilicity and Improves Proliferation and Differentiation of Human Bone Marrow Stromal Cells.

Titanium alloy (Ti6Al4V) is one of the most prominent biomaterials for bone contact because of its ability to bear mechanical loading and resist corrosion. The success of Ti6Al4V implants depends on bone formation on the implant surface. Hence, implant coatings which promote adhesion, proliferation and differentiation of bone-forming cells are desirable. One coating strategy is by adsorption of biomacromolecules. In this study, Ti6Al4V substrates produced by additive manufacturing (AM) were coated with whey protein isolate (WPI) fibrils, obtained at pH 2, and heparin or tinzaparin (a low molecular weight heparin LMWH) in order to improve the proliferation and differentiation of bone-forming cells. WPI fibrils proved to be an excellent support for the growth of human bone marrow stromal cells (hBMSC). Indeed, WPI fibrils were resistant to sterilization and were stable during storage. This WPI-heparin-enriched coating, especially the LMWH, enhanced the differentiation of hBMSC by increasing tissue non-specific alkaline phosphatase (TNAP) activity. Finally, the coating increased the hydrophilicity of the material. The results confirmed that WPI fibrils are an excellent biomaterial which can be used for biomedical coatings, as they are easily modifiable and resistant to heat treatments. Indeed, the already known positive effect on osteogenic integration of WPI-only coated substrates has been further enhanced by a simple adsorption procedure.

Marine-Inspired Enzymatic Mineralization of Dairy-Derived Whey Protein Isolate (WPI) Hydrogels for Bone Tissue Regeneration.

Whey protein isolate (WPI) is a by-product from the production of cheese and Greek yoghurt comprising ß-lactoglobulin (ß-lg) (75%). Hydrogels can be produced from WPI solutions through heating; hydrogels can be sterilized by autoclaving. WPI hydrogels have shown cytocompatibility and ability to enhance proliferation and osteogenic differentiation of bone-forming cells. Hence, they have promise in the area of bone tissue regeneration. In contrast to commonly used ceramic minerals for bone regeneration, a major advantage of hydrogels is the ease of their modification by incorporating biologically active substances such as enzymes. Calcium carbonate (CaCO3) is the main inorganic component of the exoskeletons of marine invertebrates. Two polymorphs of CaCO3, calcite and aragonite, have shown the ability to promote bone regeneration. Other authors have reported that the addition of magnesium to inorganic phases has a beneficial effect on bone-forming cell growth. In this study, we employed a biomimetic, marine-inspired approach to mineralize WPI hydrogels with an inorganic phase consisting of CaCO3 (mainly calcite) and CaCO3 enriched with magnesium using the calcifying enzyme urease. The novelty of this study lies in both the enzymatic mineralization of WPI hydrogels and enrichment of the mineral with magnesium. Calcium was incorporated into the mineral formed to a greater extent than magnesium. Increasing the concentration of magnesium in the mineralization medium led to a reduction in the amount and crystallinity of the mineral formed. Biological studies revealed that mineralized and unmineralized hydrogels were not cytotoxic and promoted cell viability to comparable extents (approximately 74% of standard tissue culture polystyrene). The presence of magnesium in the mineral formed had no adverse effect on cell viability. In short, WPI hydrogels, both unmineralized and mineralized with CaCO3 and magnesium-enriched CaCO3, show potential as biomaterials for bone regeneration.

Phenolic-Enriched Collagen Fibrillar Coatings on Titanium Alloy to Promote Osteogenic Differentiation and Reduce Inflammation.

The adsorption of biomolecules on biomaterial surfaces can promote their integration with surrounding tissue without changing their bulk properties. For biomaterials in bone reconstruction, the promotion of osteogenic differentiation and reduction of inflammation are desirable. Fibrillar coatings are interesting because of fibrils' high surface area-volume ratio, aiding adsorption and adhesion. Fibrils also serve as a matrix for the immobilization of biomolecules with biological activity, such as the phenolic compound phloroglucinol (PG), the subunit of marine polyphenols. The aim of this work was to investigate the influence of PG coatings on fibroblast- and osteoblast-like cells to increase the osseointegration of titanium implants. Collagen fibril coatings, containing PG at low and high concentrations, were produced on titanium alloy (Ti6Al4V) scaffolds generated by additive manufacturing (AM). These coatings, especially PG-enriched coatings, reduced hydrophobicity and modulated the behavior of human osteosarcoma SaOS-2 and mouse embryonic fibroblast 3T3 cell lines. Both osteoblastic and fibroblastic cells spread and adhered well on PG-enriched coatings. Coatings significantly reduced the inflammatory response. Moreover, osteogenic differentiation was promoted by collagen coatings with a high PG concentration. Thus, the enrichment of collagen fibril coatings with PG is a promising strategy to improve Ti6Al4V implants for bone contact in orthopedics and dentistry and is worthy of further investigation.

Enhancement of Biomimetic Enzymatic Mineralization of Gellan Gum Polysaccharide Hydrogels by Plant-Derived Gallotannins.

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.

Electrospun polycaprolactone membranes with Zn-doped bioglass for nasal tissues treatment.

In this work, composite membranes were investigated as future components of a layered implant for the reconstruction of nasal septum. Incorporation of zinc ions into nasal implants could potentially provide antibacterial properties to decrease or eliminate bacterial infections and subsequent surgical complications. Two types of membranes were prepared using an electrospinning method: PCL with bioglass and PCL with bioglass doped with Zn. The aim of this work was to investigate the influence of bioglass addition on the morphology, fiber diameter and composition of the membranes. The apatite-forming ability was examined in Simulated Body Fluid (SBF). The cytotoxicity of the membranes, ALP activity and in vitro mineralization were evaluated in cell culture. The mineralization and ALP activity was higher for polycaprolactone membranes modified with Zn doped bioglass than compared to pure PCL membranes or control material. The results proved that the presence of Zn2+ in the electrospun membranes = influence the osteogenic differentiation of cells.

Development of Composite Poly(Lactide-co-Glycolide)- Nanodiamond Scaffolds for Bone Cell Growth.

There are relatively few nanotechnologies that can produce nanocomposite scaffolds for cell growth. Electrospinning has emerged as the foremost method of producing nanofibrous biomimetic scaffolds for tissue engineering applications. In this study diamond nanoparticles were integrated into a polymer solution to develop a nanocomposite scaffold containing poly(lactide-co-glycolide) (PLGA) loaded with diamond nanoparticles. To investigate the effect of adding diamond nanoparticles to PLGA scaffolds, primary human mesenchymal stem cells (hMSCs) were seeded on the scaffolds. The cytocompatibility results showed that addition of diamond nanoparticles did not impinge upon cell proliferation, nor was there a cytotoxic cellular response after 9 days in culture. Scanning electron microscopy, transmission electron microscopy, atomic force microscopy and confocal microscopy enabled qualitative characterization of the fibres and revealed cell morphology and number. Furthermore, surface roughness was measured to evaluate diamond nanoparticle modifications, and no significant difference was found between the diamond nanocomposite and pure polymer scaffolds. On the other hand, bright spots on phase images performed by atomic force microscopy suggested a higher hardness at certain points on fibers of the PLGA-nanodiamond composites, which was supported by nanoindentation measurements. This study shows that PLGA nanofibers can be reinforced with nanodiamond without adversely affecting cell behaviour, and thus it sets the foundation for future application of these scaffolds in bone tissue engineering.

Comparison of platelet rich fibrin and collagen as osteoblast-seeded scaffolds for bone tissue engineering applications.

OBJECTIVES: The loss of jaw bone caused by different kinds of pathologies leads to dysfunction and reduced quality of life in affected patients. Thus, the pivotal goal in bone tissue engineering is to reconstruct these defects. The essential precondition for new tissue generation is an extracellular matrix which acts as a scaffold so that cells can migrate, differentiate, and proliferate. Fibrin, a biopolymer responsible for blood clot formation, has been shown to be suitable for tissue engineering applications. The aim of the present study is a comparison of platelet rich fibrin (PRF) with the commonly used collagen membrane BioGide(®) as a scaffold for human osteoblast cell seeding for bone tissue engineering. MATERIAL AND METHODS: Human osteoblasts were cultured with eluates from PRF (n = 7) and BioGide(®) (n = 8) membranes incubated in serum-free cell culture medium. Vitality of these cells was assessed by fluorescein diacetate and propidium iodide staining, biocompatibility with the lactate dehydrogenase test and proliferation levels with the MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide]), and BrdU (5-bromo-2-deoxyuridine) tests. In addition, human osteoblasts were seeded on both membrane systems and cell growth was compared by the water soluble tetrazolium (WST-1) (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) test and scanning electron microscopy (SEM). Osteoblastic differentiation was assessed by alkaline phosphatase activity measured by ELISA in the supernatant of osteoblasts cultivated on PRF membranes (n = 10), PRF clots (n = 10), and BioGide(®) membranes (n = 10). RESULTS: Lactate dehydrogenase test values were higher for PRF compared to BioGide(®) . The BrdU test showed superior cell growth after cultivation in eluate from PRF than in eluate from BioGide(®) . The WST-1 assay demonstrated superior cell proliferation on PRF than on BioGide(®) . SEM revealed osteoblast colonization of both membranes. Cultivation of osteoblasts on PRF membranes and PRF clots showed significantly higher alkaline phosphatase activity than on BioGide(®) membranes. CONCLUSION: Metabolic activity and proliferation of human osteoblast cells in vitro were supported to a significant higher extent by eluates from PRF membranes. Both membranes are suitable as scaffolds for cultivation of human osteoblast cells in vitro; proliferation was significant higher on PRF membranes and on PRF clot than on BioGide(®) membranes.

Designing of gradient scaffolds and their applications in tissue regeneration.

Gradient scaffolds are isotropic/anisotropic three-dimensional structures with gradual transitions in geometry, density, porosity, stiffness, etc., that mimic the biological extracellular matrix. The gradient structures in biological tissues play a major role in various functional and metabolic activities in the body. The designing of gradients in the scaffold can overcome the current challenges in the clinic compared to conventional scaffolds by exhibiting excellent penetration capacity for nutrients & cells, increased cellular adhesion, cell viability & differentiation, improved mechanical stability, and biocompatibility. In this review, the recent advancements in designing gradient scaffolds with desired biomimetic properties, and their implication in tissue regeneration applications have been briefly explained. Furthermore, the gradients in native tissues such as bone, cartilage, neuron, cardiovascular, skin and their specific utility in tissue regeneration have been discussed in detail. The insights from such advances using gradient-based scaffolds can widen the horizon for using gradient biomaterials in tissue regeneration applications.

Could Curdlan/Whey Protein Isolate/Hydroxyapatite Biomaterials Be Considered as Promising Bone Scaffolds?-Fabrication, Characterization, and Evaluation of Cytocompatibility towards Osteoblast Cells In Vitro.

The number of bone fractures and cracks requiring surgical interventions increases every year; hence, there is a huge need to develop new potential bone scaffolds for bone regeneration. The goal of this study was to gain knowledge about the basic properties of novel curdlan/whey protein isolate/hydroxyapatite biomaterials in the context of their use in bone tissue engineering. The purpose of this research was also to determine whether the concentration of whey protein isolate in scaffolds has an influence on their properties. Thus, two biomaterials differing in the concentration of whey protein isolate (i.e., 25 wt.% and 35 wt.%; hereafter called Cur_WPI25_HAp and Cur_WPI35_HAp, respectively) were fabricated and subjected to evaluation of porosity, mechanical properties, swelling ability, protein release capacity, enzymatic biodegradability, bioactivity, and cytocompatibility towards osteoblasts in vitro. It was found that both biomaterials fulfilled a number of requirements for bone scaffolds, as they demonstrated limited swelling and the ability to undergo controllable enzymatic biodegradation, to form apatite layers on their surfaces and to support the viability, growth, proliferation, and differentiation of osteoblasts. On the other hand, the biomaterials were characterized by low open porosity, which may hinder the penetration of cells though their structure. Moreover, they had low mechanical properties compared to natural bone, which limits their use to filling of bone defects in non-load bearing implantation areas, e.g., in the craniofacial area, but then they will be additionally supported by application of mechanically strong materials such as titanium plates. Thus, this preliminary in vitro research indicates that biomaterials composed of curdlan, whey protein isolate, and hydroxyapatite seem promising for bone tissue engineering applications, but their porosity and mechanical properties should be improved. This will be the subject of our further work.

Freeze-Dried Curdlan/Whey Protein Isolate-Based Biomaterial as Promising Scaffold for Matrix-Associated Autologous Chondrocyte Transplantation-A Pilot In-Vitro Study.

The purpose of this pilot study was to establish whether a novel freeze-dried curdlan/whey protein isolate-based biomaterial may be taken into consideration as a potential scaffold for matrix-associated autologous chondrocyte transplantation. For this reason, this biomaterial was initially characterized by the visualization of its micro- and macrostructures as well as evaluation of its mechanical stability, and its ability to undergo enzymatic degradation in vitro. Subsequently, the cytocompatibility of the biomaterial towards human chondrocytes (isolated from an orthopaedic patient) was assessed. It was demonstrated that the novel freeze-dried curdlan/whey protein isolate-based biomaterial possessed a porous structure and a Young's modulus close to those of the superficial and middle zones of cartilage. It also exhibited controllable degradability in collagenase II solution over nine weeks. Most importantly, this biomaterial supported the viability and proliferation of human chondrocytes, which maintained their characteristic phenotype. Moreover, quantitative reverse transcription PCR analysis and confocal microscope observations revealed that the biomaterial may protect chondrocytes from dedifferentiation towards fibroblast-like cells during 12-day culture. Thus, in conclusion, this pilot study demonstrated that novel freeze-dried curdlan/whey protein isolate-based biomaterial may be considered as a potential scaffold for matrix-associated autologous chondrocyte transplantation.

Biomimetic biphasic curdlan-based scaffold for osteochondral tissue engineering applications - Characterization and preliminary evaluation of mesenchymal stem cell response in vitro.

Osteochondral defects remain a huge problem in medicine today. Biomimetic bi- or multi-phasic scaffolds constitute a very promising alternative to osteochondral autografts and allografts. In this study, a new curdlan-based scaffold was designed for osteochondral tissue engineering applications. To achieve biomimetic properties, it was enriched with a protein component - whey protein isolate as well as a ceramic ingredient - hydroxyapatite granules. The scaffold was fabricated via a simple and cost-efficient method, which represents a significant advantage. Importantly, this technique allowed generation of a scaffold with two distinct, but integrated phases. Scanning electron microcopy and optical profilometry observations demonstrated that phases of biomaterial possessed different structural properties. The top layer of the biomaterial (mimicking the cartilage) was smoother than the bottom one (mimicking the subchondral bone), which is beneficial from a biological point of view because unlike bone, cartilage is a smooth tissue. Moreover, mechanical testing showed that the top layer of the biomaterial had mechanical properties close to those of natural cartilage. Although the mechanical properties of the bottom layer of scaffold were lower than those of the subchondral bone, it was still higher than in many analogous systems. Most importantly, cell culture experiments indicated that the biomaterial possessed high cytocompatibility towards adipose tissue-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells in vitro. Both phases of the scaffold enhanced cell adhesion, proliferation, and chondrogenic differentiation of stem cells (revealing its chondroinductive properties in vitro) as well as osteogenic differentiation of these cells (revealing its osteoinductive properties in vitro). Given all features of the novel curdlan-based scaffold, it is worth noting that it may be considered as promising candidate for osteochondral tissue engineering applications.

The influence of Ca/Mg ratio on autogelation of hydrogel biomaterials with bioceramic compounds.

Hydrogels, which are versatile three-dimensional structures containing polymers and water, are very attractive for use in biomedical fields, but they suffer from rather weak mechanical properties. In this regard, biocompatible particles can be used to enhance their mechanical properties. The possibility of loading such particles with drugs (e.g. enzymes) makes them a particularly useful component in hydrogels. In this study, micro/nanoparticles containing various ratios of Ca2+/Mg2+ with sizes ranging from 1 to 8 µm were prepared and mixed with gellan gum (GG) solution to study the in-situ formation of hydrogel-particle composites. The particles provide multiple functionalities: 1) they efficiently crosslink GG to induce hydrogel formation through the release of the divalent cations (Ca2+/Mg2+) known to bind to GG polymer chains; 2) they enhance mechanical properties of the hydrogel from 2 up to 100 kPa; 3) the samples most efficiently promoting cell growth were found to contain two types of minerals: vaterite and hydroxymagnesite, which enhanced cells proliferation and hydroxyapatite formation. The results demonstrate that such composite materials are attractive candidates for applications in bone regeneration.

Comparison of in vitro biocompatibility of NanoBone(®) and BioOss(®) for human osteoblasts.

INTRODUCTION: Scaffolds for bone tissue engineering seeded with the patient's own cells might be used as a preferable method to repair bone defects in the future. With the emerging new technologies of nanostructure design, new synthetic biomaterials are appearing on the market. Such scaffolds must be tested in vitro for their biocompatibility before clinical application. However, the choice between a natural or a synthetic biomaterial might be challenging for the doctor and the patient. In this study, we compared the biocompatibility of a synthetic bone substitute, NanoBone(®) , to the widely used natural bovine bone replacement material BioOss(®) . MATERIAL AND METHODS: The in vitro behaviour of human osteoblasts on both materials was investigated. Cell performance was determined using scanning electron microscopy (SEM), cell vitality staining and four biocompatibility tests (LDH, MTT, WST, BrdU). RESULTS: We found that both materials showed low cytotoxicity and good biocompatibility. The MTT proliferation test was superior for Nanobone(®) . DISCUSSION: Both scaffolds caused only little damage to human osteoblasts and justify their clinical application. However, NanoBone(®) was able to support and promote proliferation of human osteoblasts slightly better than BioOss(®) in our chosen test set-up. The results may guide doctors and patients when being challenged with the choice between a natural or a synthetic biomaterial. Further experiments are necessary to determine the comparison of biocompatibility in vivo.

Phloroglucinol-enhanced whey protein isolate hydrogels with antimicrobial activity for tissue engineering.

Aging populations in developed countries will increase the demand for implantable materials to support tissue regeneration. Whey Protein Isolate (WPI), derived from dairy industry by-products, can be processed into hydrogels with the following desirable properties for applications in tissue engineering: (i) ability to support adhesion and growth of cells; (ii) ease of sterilization by autoclaving and (iii) ease of incorporation of poorly water-soluble drugs with antimicrobial activity, such as phloroglucinol (PG), the fundamental phenolic subunit of marine polyphenols. In this study, WPI hydrogels were enriched with PG at concentrations between 0 and 20% w/v. PG solubilization in WPI hydrogels is far higher than in water. Enrichment with PG did not adversely affect mechanical properties, and endowed antimicrobial activity against a range of bacteria which occur in healthcare-associated infections (HAI). WPI-PG hydrogels supported the growth of, and collagen production by human dental pulp stem cells and - to a lesser extent - of osteosarcoma-derived MG-63 cells. In summary, enrichment of WPI with PG may be a promising strategy to prevent microbial contamination while still promoting stem cell attachment and growth.

Surface functionalization of chitosan as a coating material for orthopaedic applications: A comprehensive review.

Metallic implants have dominated the biomedical implant industries for the past century for load-bearing applications, while the polymeric implants have shown great promise for tissue engineering applications. The surface properties of such implants are critical as the interaction of implant surfaces, and the body tissues may lead to unfavourable reactions. Desired implant properties are biocompatibility, corrosion resistance, and antibacterial activity. A polymer coating is an efficient and economical way to produce such surfaces. A lot of research has been carried out on chitosan (CS)-modified metallic and polymer scaffolds in the last decade. Different methods such as electrophoretic deposition, sol-gel methods, dip coating and spin coating, electrospinning, etc. have been utilized to produce CS coatings. However, a systematic review of chitosan coatings on scaffolds focussing on widely employed techniques is lacking. This review surveys literature concerning the current status of orthopaedic applications of CS for the purpose of coatings. In this review, the various preparation methods of coating, and the role of the surface functionalities in determining the efficiency of coatings are discussed. Effect of nanoparticle additions on the polymeric interfaces and in regulating the properties of surface coatings are also investigated in detail.

Novel ceramic bone replacement material CeraBall seeded with human mesenchymal stem cells.

OBJECTIVES: Hydroxyapatite (HA) and tricalcium phosphate (TCP) are two very common ceramic materials for bone replacement. A recently developed material for bone replacement is CeraBall, which is a mixed HA-TCP scaffold available as porous spherical scaffolds of diameter 4 and 6 mm. Before their use as bone replacement materials in vivo, in vitro testing of these scaffolds is necessary. The goal of this study was to characterise 4 and 6 mm CeraBall scaffolds in vitro with a view to their future use as bone replacement materials. MATERIALS AND METHODS: The proliferation of human mesenchymal stromal cells (hMSCs) seeded on CeraBall scaffolds was evaluated quantitatively using the WST [Water soluble tetrazolium ((4-[3-(4- Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate)] test and qualitatively by scanning electron microscopy (SEM). In addition, the standard MTT [(3-(4, 5-Dimenthylthiazol-2-Y1)-2, 5-Diphenyltetrazolium bromide)] biocompatibility test and cell vitality staining were performed using hMSCs. CeraBall scaffolds were also tested for their mechanical properties. RESULTS: SEM and WST test results showed that hMSCs proliferated on CeraBall scaffolds over the course of 9 days. Proliferation was similar to that seen on tissue culture polystyrene (control). Cells showed a well-spread morphology and formed 'sheets' on the surface of scaffolds. Invasion of pores was observed. Good biocompatibility was demonstrated by MTT test results and cell vitality staining. Scaffolds of both 4 and 6 mm were able to withstand compressive loads of 5 N. CONCLUSIONS: CeraBall scaffolds show good biocompatibility in vitro for hMSCs. This opens the way for in vivo applications.

Platelet-rich fibrin membranes as scaffolds for periosteal tissue engineering.

OBJECTIVES: Platelet-rich fibrin (PRF)-based membranes have been used for covering alveolar ridge augmentation side in several in vivo studies. Few in vitro studies on PRF and no studies using human periosteal cells for tissue engineering have been published. The aim is a comparison of PRF with the commonly used collagen membrane Bio-Gide as scaffolds for periosteal tissue engineering. MATERIAL AND METHODS: Human periosteal cells were seeded on membrane pieces (collagen [Bio-Gide] and PRF) at a density of 10(4) cells/well. Cell vitality was assessed by fluorescein diacetate (FDA) and propidium iodide (PI) staining, biocompatibility with the lactate dehydrogenase (LDH) test and proliferation level with the MTT, WST and BrdU tests and scanning electron microscopy (SEM). RESULTS: PRF membranes showed slightly inferior biocompatibility, as shown by the LDH test. The metabolic activity measured by the MTT and WST tests was higher for PRF than for collagen (BioGide). The proliferation level as measured by the BrdU test (quantitative) and SEM examinations (qualitative) revealed higher values for PRF. CONCLUSION: PRF appears to be superior to collagen (Bio-Gide) as a scaffold for human periosteal cell proliferation. PRF membranes are suitable for in vitro cultivation of periosteal cells for bone tissue engineering.

Hydrostatic pressure stimulation of human mesenchymal stem cells seeded on collagen-based artificial extracellular matrices.

Human mesenchymal stem cells (hMSCs) from bone marrow are considered a promising cell source for bone tissue engineering applications because of their ability to differentiate into cells of the osteoblastic lineage. Mechanical stimulation is able to promote osteogenic differentiation of hMSC; however, the use of hydrostatic pressure (HP) has not been well studied. Artificial extracellular matrices containing collagen and chondroitin sulfate (CS) have promoted the expression of an osteoblastic phenotype by hMSCs. However, there has been little research into the combined effects of biochemical stimulation by matrices and simultaneous mechanical stimulation. In this study, artificial extracellular matrices generated from collagen and/or CS were coated onto polycaprolactone-co-lactide substrates, seeded with hMSCs and subjected to cyclic HP at various time points during 21 days after cell seeding to investigate the effects of biochemical, mechanical, and combined biochemical and mechanical stimulations. Cell differentiation was assessed by analyzing the expression of alkaline phosphatase (ALP) at the protein- and mRNA levels, as well as for calcium accumulation. The timing of HP stimulation affected hMSC proliferation and expression of ALP activity. HP stimulation after 6 days was most effective at promoting ALP activity. CS-containing matrices promoted the osteogenic differentiation of hMSCs. A combination of both CS-containing matrices and cyclic HP yields optimal effects on osteogenic differentiation of hMSCs on scaffolds compared with individual responses.

Glucuronic acid and phosphoserine act as mineralization mediators of collagen I based biomimetic substrates.

Glucuronic acid (GlcA) and phosphoserine (pS) carrying acidic functional groups were used as model molecules for glycosaminoglycans and phosphoproteins, respectively to mimic effects of native biomolecules and influence the mineralization behaviour of collagen I. Collagen substrates modified with GlcA showed a stable interaction between GlcA and collagen fibrils. Substrates were mineralized using the electrochemically assisted deposition (ECAD) in a Ca(2+)/H( x )PO (4) ((3-x)) electrolyte at physiological pH and temperature. During mineralization of collagen-GlcA matrices, crystalline hydroxyapatite (HA) formed earlier with increasing GlcA content of the collagen matrix, while the addition of pS to the electrolyte succeeded in inhibiting the transformation of preformed amorphous calcium phosphate (ACP) to HA. The lower density of the resulting mineralization and the coalesced aggregates formed at a certain pS concentration suggest an interaction between calcium and the phosphate groups of pS involving the formation of complexes. Combining GlcA-modified collagen and pS-modified electrolyte showed dose-dependent cooperative effects.

Porous polymer/hydroxyapatite scaffolds: characterization and biocompatibility investigations.

Poly-lactic-glycolic acid (PLGA) has been widely used as a scaffold material for bone tissue engineering applications. 3D sponge-like porous scaffolds have previously been generated through a solvent casting and salt leaching technique. In this study, polymer-ceramic composite scaffolds were created by immersing PLGA scaffolds in simulated body fluid, leading to the formation of a hydroxyapatite (HAP) coating. The presence of a HAP layer was confirmed using scanning electron microscopy, energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy in attenuated total reflection mode. HAP-coated PLGA scaffolds were tested for their biocompatibility in vitro using human osteoblast cell cultures. Biocompatibility was assessed by standard tests for cell proliferation (MTT, WST), as well as fluorescence microscopy after standard cell vitality staining procedures. It was shown that PLGA-HAP composites support osteoblast growth and vitality, paving the way for applications as bone tissue engineering scaffolds.