Enhancement of osteogenesis by poly(lactide-co-glycolide) sponges loaded with surface-embedded hydroxyapatite particles and rhBMP-2

The bone mesenchymal stem cells (BMSCs) were seeded on [poly(lactide-co-glycolide) scaffolds with hydroxyapatite (HA) coating, and "s" stands for surface] (PLGA/HA-S), PLGA/HA-M (containing the same HA amount in the matrix as that of the PLGA/HA-S and "m" stands for matrix), and PLGA scaffolds, which were then cultured in a medium-containing Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2). In vitro culture of rat BMSCs found no different cell morphology in all the scaffolds, but the alkaline phosphatase activity and osteogenic gene expression of type I collagen (COL I) and osteocalcin (OCN) in the PLGA/HA-S scaffolds were always highest and were significantly improved in comparison with those in the PLGA scaffolds. In a rat calvarial defect model, new bone formation was enhanced in the PLGA/HA-S/ErhBMP-2 implants at 4 and 8 weeks after implantation too. Therefore, the PLGA/HA-S scaffold can better enhance the ErhBMP-2-induced osteogenic differentiation of BMSCs in vitro and osteogenesis in vivo.     

Compressive properties of polymer coated synthetic hydroxyapatite for bone grafting

A porous hydroxyapatite material hydrothermally converted from the calcium carbonate exoskeleton of the coral, genus Goniopora (CHAG) was either microcoated using polymethylmethacrylate (PMMA) or polylactic acid (PLA) to cover all internal surfaces, or externally coated to produce a shell, with the objective of reducing the brittleness of the material. Compressive testing showed that while CHAG, externally coated with PMMA, showed the largest increases in strength, stiffness, and energy absorption, the uncoated hydroxyapatite core cracked at low loads although the shell remained intact. CHAG internally microcoated with PMMA showed a 3.84 increase in compressive strength while specimens with internal PLA coatings had 1.81 times the compressive strength of uncoated CHAG specimens. Compared to the mechanical properties of cancellous graft material, specimens internally microcoated with either polymer could be produced having properties equivalent to or greater than those of cancellous graft.     

Enhancement of in vitro osteogenesis on titanium by chemically produced nanotopography

The surface characteristics of biomaterials can influence protein adsorption, cellular functions, and ultimately tissue formation. Controlled chemical oxidation of titanium-based surfaces with a mixture of H(2)SO(4)/H(2)O(2) creates a nanopatterned surface that has been shown to affect early osteogenic events. The objective of this study was to evaluate the effect over time of this nanopattern on various key parameters of osteogenesis, and determine whether these effects ultimately translate into more mineralized matrix production. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvaria and grown on treated and untreated titanium discs for periods of up to 14 days. Alkaline phosphatase activity peaked earlier and cell number was higher as of day 7 on the nanopatterned discs. Immunofluorescence showed that the treated surface favored early bone sialoprotein and osteopontin secretion, and fibronectin accumulation. Alizarin red staining revealed that, at days 10 and 14, there were significantly more mineralized nodules on treated than on untreated discs. These results demonstrate that simple chemical treatment of titanium with H(2)SO(4)/H(2)O(2) accelerates the in vitro osteogenic potential of calvaria-derived cells. They also suggest that this treatment may represent an advantageous approach for producing "intelligent surfaces" that stimulate bone formation and enhance bone-implant contact.     

The in vitro corneal biocompatibility of hydroxyapatite coated carbon mesh

The purpose of this study was to consider the use of a hydroxyapatite (HA) coated porous carbon matrix as a synthetic dental laminate substitute in osteo-odonto-keratoprosthetic (OOKP) design. 3 types of carbon meshes were coated with HA by sonoelectrochemical deposition. The materials were characterised by scanning electron microscopy (SEM) and HA deposition was characterised by elemental analysis and X-ray diffractometry (XRD). In vitro assays were carried out to quantify the effects of HA coating on human keratocyte adhesion. Cellular cytokine production was used to assess inflammatory potential. HA coating significantly increased keratocyte adhesion to the carbon matrix (p < 0.01). The materials did not induce excessive cytokine production by the adherent keratocytes. In addition, the matrices themselves adsorbed significant levels of the cytokine IL-8 (p < 0.05). The results indicate that HA coated carbon matrices provide a suitable environment to enhance in-growth of corneal cells without inducing further inflammation. The materials may also suppress excessive inflammation by adsorption of the cytokine IL-8 into the porous, internal carbon structure.     

Bone-bonding ability of a hydroxyapatite coated zirconia-alumina nanocomposite with a microporous surface

Using a combination of hydroxyapatite (HA) coating and microporous surface treatment, bone-bonding ability was given to composites of ceria-stabilized tetragonal zirconia and alumina (CZA), which possesses excellent mechanical and wear properties and phase stability. Four types of CZA plates (2 x 10 x 15 mm3) were prepared for this study, which were CZA with a polished surface (group 1), a microporous surface prepared by hydrofluoric acid and heat treatment (group 2), a microporous surface with a submicron HA coating prepared by alternately soaking the plate from group 2 in aqueous CaCl2/HCl and Na2HPO4 solutions (group 3), and a microporous surface with a 4-microm HA coating prepared by the biomimetic method, where the plates from group 3 were soaked in simulated body fluid (group 4). Plates were implanted into rabbit tibia, and after 4, 8, and 16 weeks, tensile testing and histological examination of the bone-implant interface were conducted. At 4 weeks, group 4 had superior bone-bonding ability compared with other implants, which was maintained at the later postimplantation times. This HA-coated CZA with a microporous surface has the possibility of clinical use as a bearing material in cementless joint prostheses or as a load-bearing bone substitute.     

Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) supports in vitro osteogenesis

Studies have demonstrated that polymeric biomaterials have the potential to support osteoblast growth and development for bone tissue repair. Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV), a bioabsorbable, biocompatible polyhydroxy acid polymer, is an excellent candidate that, as yet, has not been extensively investigated for this purpose. As such, we examined the attachment characteristics, self-renewal capacity, and osteogenic potential of osteoblast-like cells (MC3T3-E1 S14) when cultured on PHBV films compared with tissue culture polystyrene (TCP). Cells were assayed over 2 weeks and examined for changes in morphology, attachment, number and proliferation status, alkaline phosphatase (ALP) activity, calcium accumulation, nodule formation, and the expression of osteogenic genes. We found that these spindle-shaped MC3T3-E1 S14 cells made cell-cell and cell-substrate contact. Time-dependent cell attachment was shown to be accelerated on PHBV compared with collagen and laminin, but delayed compared with TCP and fibronectin. Cell number and the expression of ALP, osteopontin, and pro-collagen alpha1(I) mRNA were comparable for cells grown on PHBV and TCP, with all these markers increasing over time. This demonstrates the ability of PHBV to support osteoblast cell function. However, a lag was observed for cells on PHBV in comparison with those on TCP for proliferation, ALP activity, and cbfa-1 mRNA expression. In addition, we observed a reduction in total calcium accumulation, nodule formation, and osteocalcin mRNA expression. It is possible that this cellular response is a consequence of the contrasting surface properties of PHBV and TCP. The PHBV substrate used was rougher and more hydrophobic than TCP. Although further substrate analysis is required, we conclude that this polymer is a suitable candidate for the continued development as a biomaterial for bone tissue engineering.     

A synthetic peptide with hepatitis B surface antigen reactivity

A computerized analysis of the amino acid sequence of the hepatitis B surface antigen protein was used to predict the location of an antigenic determinant in a limited portion of the molecule, comprising residues 138 through 149. This sequence was synthesized by the Merrifield procedure and assayed for its ability to bind HBsAg antibodies. The peptide bound up to 9% of antibodies directed to a mixture of ad and ay viral subtypes. The shape of the curve obtained indicates that the peptide may be capable of binding a much greater percentage of such antibodies. The peptide therefore represents a significant antigenic determinant of HBsAg.     

Enhancement of peptide coupling to hydroxyapatite and implant osseointegration through collagen mimetic peptide modified with a polyglutamate domain

Hydroxyapatite (HA) is a widely-used biomaterial for bone repair due to its high degree of osteoconductivity. However, strategies for improving HA performance by functionalizing surfaces with bioactive factors are limited. In this study, we explored the use of a HA-binding domain (heptaglutamate, "E7") to facilitate coupling of the collagen mimetic peptide, DGEA, to two types of HA-containing materials, solid HA disks and electrospun polycaprolactone matrices incorporating nanoparticulate HA. We found that the E7 domain directed significantly more peptide to the surface of HA and enhanced peptide retention on both materials in vitro. Moreover, E7-modified peptides were retained in vivo for at least two months, highlighting the potential of this mechanism as a sustained delivery system for bioactive peptides. Most importantly, E7-DGEA-coupled HA, as compared with DGEA-HA, enhanced the adhesion and osteoblastic differentiation of mesenchymal stem cells, and also increased new bone formation and direct bone-implant contact on HA disks implanted into rat tibiae. Collectively, these results support the use of E7-DGEA peptides to promote osteogenesis on HA substrates, and further suggest that the E7 domain can serve as a universal tool for anchoring a wide variety of bone regenerative molecules to any type of HA-containing material.     

Enhancement of viral hepatitis B antibody (Anti-HBs) response to a synthetic cyclic peptide by priming with anti-idiotype antibodies

In vivo injections of anti-idiotype antibodies were used to prime the immune system of mice to hepatitis B surface antigen (HBsAg). Anti-idiotype reagents in conjunction with a cyclic synthetic peptide analogous to positions 122-137 of HBsAg induced an antibody response to HBsAg (anti-HBs) comparable to that obtained with a single injection of intact HBsAg particles. In addition, high anti-HBs titers were produced in mice injected with HBsAg following anti-idiotype priming. These data indicate that anti-idiotype antibodies may be useful in priming the immune system of a host to a potential infectious agent.     

In vitro osteogenesis on a highly bioactive glass-ceramic (Biosilicate)

One of the strategies to improve the mechanical performance of bioactive glasses for load-bearing implant devices has been the development of glass-ceramic materials. The present study aimed to evaluate the effect of a highly bioactive, fully-crystallized glass-ceramic (Biosilicate) of the system P(2)O(5)-Na(2)O-CaO-SiO(2) on various key parameters of in vitro osteogenesis. Surface characterization was carried out by scanning electron microscopy and Fourier transform infrared spectroscopy. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvarial bone and by growing on Biosilicate discs and on control bioactive glass surfaces (Biosilicate) parent glass and Bioglass(R) 45S5) for periods of up to 17 days. All materials developed an apatite layer in simulated body fluid for 24h. Additionally, as early as 12 h under culture conditions and in the absence of cells, all surfaces developed a layer of silica-gel that was gradually covered by amorphous calcium phosphate deposits, which remained amorphous up to 72 h. During the proliferative phase of osteogenic cultures, the majority of cells exhibited disassembly of the actin cytoskeleton, whereas reassembly of actin stress fibers took place only in areas of cell multilayering by day 5. Although no significant differences were detected in terms of total protein content and alkaline phosphatase activity at days 11 and 17, Biosilicate supported significantly larger areas of calcified matrix at day 17. The results indicate that full crystallization of bioactive glasses in a range of compositions of the system P(2)O(5)-Na(2)O-CaO-SiO(2) may promote enhancement of in vitro bone-like tissue formation in an osteogenic cell culture system.     

In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with poly(L-lactide)

Nanocomposite of hydroxyapatite (HAP) surface-grafted with poly(l-lactide) (PLLA) (g-HAP) shows a wide application for bone fixation materials due to its improved interface compatibility, mechanical property and biocompatibility in our previous study. In this paper, a 3-D porous scaffold of g-HAP/poly(lactide-co-glycolide) (PLGA) was fabricated using the solvent casting/particulate leaching method to investigate its applications in bone replacement and tissue engineering. The composite of un-grafted HAP/PLGA and neat PLGA were used as controls. Their in vivo mineralization and osteogenesis were investigated by intramuscular implantation and replacement for repairing radius defects of rabbits. After surface modification, more uniform distribution of g-HAP particles but a lower calcium exposure on the surface of g-HAP/PLGA was observed. Intramuscular implantation study showed that the scaffold of g-HAP/PLGA was more stable than that of PLGA, and exhibited similar mineralization and biodegradability to HAP/PLGA at the 12-20 weeks post-surgery. The implantation study for repairing critical radius defects showed that the scaffold of g-HAP/PLGA exhibited rapid and strong mineralization and osteoconductivity, and the incorporation of BMP-2 could enhance the osteogenic process of the composite implant. The new bone formation with the intact structure of a long bone was guided by the implant of g-HAP/PLGA.     

Bonding osteogenesis in coralline hydroxyapatite combined with bone marrow cells

Coralline hydroxyapatite ceramics alone (control) and the ceramics combined with rat marrow cells were implanted subcutaneously in the backs of syngeneic Fischer rats and harvested at 1,2,3,4,6,8 and 24 wk after surgery. None of the control ceramics (without marrow) showed bone formation. However, ceramics combined with marrow cells showed consistent new bone formation in the pore regions. Histometrical results revealed increased new bone formation over time. Undecalcified sections of the ceramics studied by fluorochrome labelling showed that the osteogenesis began directly on the surface of the ceramic and proceeded centripetally towards the centre of the pores (bonding osteogenesis). SEM-EPMA analysis of the bone-ceramic interface also revealed direct bonding of bone to the ceramic surface.     

Selective osteogenesis by a synthetic mineral inducing peptide for the treatment of osteoporosis

Mineralization in mammalian cells is accomplished by concerted regulation of protein-based extracellular matrix (ECM) components, such as non-collagenous proteins and collagen fibrils. In this study, we investigated the ability of a collagen-binding motif (CBM) peptide derived from osteopontin to selectively affect osteogenic or adipogenic differentiation in vitro and in vivo. In particular, increased osteogenic differentiation and decreased adipogenic differentiation were observed in human mesenchymal stem cells (hMSCs). Osteocalcin (OCN) protein expression in MC3T3-E1 cells without osteogenic inducers was then investigated following treatment with the CBM peptide. In ovariectomized (OVX) mice, estrogen deficiency induced osteoporosis and increased fat tissue deposition. However, after the CBM peptide or estradiol was injected into the OVX mice for 2 months, the increased serum OCN concentration and alkaline phosphate (ALP) activity were decreased in the estradiol-treated group (OVX-E) and the high-concentration CBM peptide-treated group (OVX-HP). Significant bone loss was also observed in the ovariectomized mice (OVX-PBS). In particular, the bone volume per total volume (BV/TV) and bone mineral density (BMD) were significantly decreased in the OVX mice; however, both of these markers were restored in the OVX-HP group, which also had significantly well-developed bone structure and bone formation. In contrast to the bone structural change, adipose tissue was increased in the OVX-PBS. However, a significant decrease in total fat and subcutaneous fat was observed in the low-concentration CBM peptide-treated group (OVX-LP) and the estradiol-treated group (OVX-E). Taken together, these results suggest that the CBM peptide could be an effective therapeutic agent for osteoporosis due to its selective stimulation of osteogenic differentiation, rather than adipogenesis.     

Inhibition of angiogenesis in vitro by Arg-Gly-Asp-containing synthetic peptide.

This study was designed to evaluate the effect of the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) on angiogenesis in serum-free collagen gel culture of rat aorta. The GRGDS peptide contains the amino acid sequence Arg-Gly-Asp (RGD), which has been implicated as a recognition site in interactions between extracellular matrix (ECM) molecules and cell membrane receptors. RGD-containing synthetic peptides are known to inhibit attachment of endothelial cells to substrates, but their effect on angiogenesis has not been fully characterized. Aortic explants embedded in collagen gel in the absence of GRGDS generated branching microvessels through a process of endothelial migration and proliferation. Addition of GRGDS to the culture medium caused a marked inhibition of angiogenesis. In contrast, GRGES, a control peptide lacking the RGD sequence, failed to inhibit angiogenesis. The inhibitory effect of GRGDS was nontoxic and reversible. The angiogenic activity of aortic explants previously inhibited with GRGDS could be restored by incubating the cultures in GRGDS-free medium. These findings suggest that angiogenesis is an anchorage-dependent process that can be inhibited by interfering with the attachment of endothelial cells to the ECM. It also indicates that synthetic peptides can be used as probes to study the mechanisms by which the ECM regulates angiogenesis.     

Mechanical and biological properties of hydroxyapatite/tricalcium phosphate scaffolds coated with poly(lactic-co-glycolic acid)

Regeneration of bone, cartilage and osteochondral tissues by tissue engineering has attracted intense attention due to its potential advantages over the traditional replacement of tissues with synthetic implants. Nevertheless, there is still a dearth of ideal or suitable scaffolds based on porous biomaterials, and the present study was undertaken to develop and evaluate a useful porous composite scaffold system. Here, hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds (average pore size: 500 μm; porosity: 87%) were prepared by a polyurethane foam replica method, followed by modification with infiltration and coating of poly(lactic-co-glycolic acid) (PLGA). The thermal shock resistance of the composite scaffolds was evaluated by measuring the compressive strength before and after quenching or freezing treatment. The porous structure (in terms of pore size, porosity and pore interconnectivity) of the composite scaffolds was examined. The penetration of the bone marrow stromal stem cells into the scaffolds and the attachment of the cells onto the scaffolds were also investigated. It was shown that the PLGA incorporation in the HA/TCP scaffolds significantly increased the compressive strength up to 660 kPa and the residual compressive strength after the freezing treatment decreased to 160 kPa, which was, however, sufficient for the scaffolds to withstand subsequent cell culture procedures and a freeze–drying process. On the other hand, the PLGA coating on the strut surfaces of the scaffolds was rather thin (<5 μm) and apparently porous, maintaining the high open porosity of the HA/TCP scaffolds, resulting in desirable migration and attachment of the bone marrow stromal stem cells, although a thicker PLGA coating would have imparted a higher compressive strength of the PLGA-coated porous HA/TCP composite scaffolds.     

Assembly of collagen-binding peptide with collagen as a bioactive scaffold for osteogenesis in vitro and in vivo

Bioactive scaffolds inducing cell adhesion, differentiation have been premise for optimal formation of target tissue. Collagen has been employed as a tissue regenerative scaffold especially for bone regeneration and has been chemically surface-modified to present bioactivity. Herein, we show that peptide, denoted as collagen-binding motif (CBM, GLRSKSKKFRRPDIQYPDATDEDITSHM) identified from osteopontin (OPN) protein, was able to specifically bind collagen without chemical conjugation, while presenting apatite forming capability in vitro and in vivo. Collagen surface alone was not able to induce noticeable apatite nucleation however, mineralization was evident when assembled with CBM peptide, implying that the collagen-CBM assembly played a pivotal role in biomineralization. In vivo result further demonstrated that the CBM peptide in complex with material was able to induce bone formation by helping mineralization in the bone defect. Taken together, the CBM peptide herein and its assembly with collagen can be applied as an inducer of biomineralization as well as a bioactive scaffold for bone regeneration.     

Monoclonal antibodies to hepatitis B surface antigen (HBsAg) with anti-alpha specificity recognize a synthetic peptide analogue (S135-155) with unmodified lysine (141)

A synthetic peptide corresponding to residues 135-155 (S135-155) of the major protein component of HBsAg was conjugated to beta-galactosidase. This conjugate reacted with monoclonal anti-HBs antibodies having anti-alpha group specificity. The reaction was inhibited by: HBsAg of either subtype ad or ay; by unconjugated S135-155 or a shorter peptide S140-155, but not by unrelated peptides. Modification of lysine residues of either HBsAg or S135-155 reduced this inhibitory effect. These results indicate that Lys 141 is essential for maintaining the antigenicity of one of the epitopes responsible for the common alpha specificity of HBsAg and that studies involving the use of synthetic peptides and modifications of distinct amino acid residues in the native protein or in the peptide may help in characterizing epitopes of viral antigens in general.     

Osteogenic differentiation of cultured marrow stromal stem cells on surface of microporous hydroxyapatite based mica composite and macroporous synthetic hydroxyapatite.

In order to investigate the significance of hydroxyapatite based microporous composite (HA/mica composite) surfaces and a macroporous synthetic hydroxyapatite, rat marrow cell culture, which shows osteogenic differentiation, was carried out on six different culture substrata (two control culture dishes, two identical HA/mica composites, and two identical macroporous synthetic hydroxyapatites). A culture period of two weeks in the presence of beta-glycerophosphate (BGP), ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain. The stain on the macroporous synthetic hydroxyapatite and the HA/mica composites were intense, the enzyme activity being about double that of control culture dishes. These data indicate that the synthetic macroporous hydroxyapatite surface and the HA/mica composite surface promotes osteoblastic differentiation.     

Characterization of human primary osteoblast response on bioactive glass (BaG 13-93)- coated poly-L,DL-lactide (SR-PLA70) surface in vitro

Bioabsorbable polylactide-based polymers are commonly used for bone reconstruction. Although these polymers have proven successful in many applications, they do not have the capacity to induce osteoconduction. Therefore, several strategies have been developed to manufacture osteoconductive polylactide-based composites. In this study, we have investigated in vitro response of human primary osteoblasts for self-reinforced poly-L,DL-lactide 70/30 (SR-PLA70) plates coated with spheres of bioactive glass 13-93 (SR-PLA70 + BaG). Osteoblasts were cultured on SR-PLA70 and SR-PLA70 + BaG plates for 2, 7, or 14 days. By day 7, both materials induced a reduction in total cell population. However, by day 14 the proliferative response of osteoblasts on SR-PLA70 + BaG surface was such that the cell population had regained similar levels as that of day 2 controls. Alkaline phosphatase activity was higher on SR-PLA70 at day 7 but declined to control levels by day 14. There were no significant time-dependent variations in alkaline phosphatase activity on SR-PLA70 + BaG. After in vitro hydrolysis for 7 days, the elemental analysis of SR-PLA70 + BaG surface showed the presence of mineral precipitates that were confirmed as crystalline hydroxyapatite. This was accompanied by osteoblast spreading, protrusions of microvilli adhered to BaG 19-39 surface, cuboidal phenotype and cell surface associated formation of hydroxyapatite microspheres. In conclusion, the SR-PLA70 + BaG composite is capable of inducing a proliferative response of human primary osteoblasts, and appears to support the development of mature osteoblast phenotype. Therefore, the SR-PLA70 + BaG composites appear as promising osteoconductive scaffold candidates for reconstruction and regeneration of bone matrix.