Juliette van den Dolder

Publications of Juliette van den Dolder

• Evaluation of an orthotopically implanted calcium phosphate cement containing gelatin microparticles.

  Authors: Dennis P Link, Juliette van den Dolder, Jeroen J J P van den Beucken, Wouter Habraken, Annemieke Soede, Otto C Boerman, Antonios G Mikos, John A Jansen

  Journal of biomedical materials research. Part A. 08/2009; 90(2):372-9.

  This study focused on the degradation properties of gelatin microparticles incorporated in calcium phosphate (CaP) cement and the subsequent effect of these composites on bone formation. PositivelyThis study focused on the degradation properties of gelatin microparticles incorporated in calcium phosphate (CaP) cement and the subsequent effect of these composites on bone formation. Positively charged alkaline gelatin (type A) microparticles or negatively charged acidic gelatin (type B) microparticles were incorporated in CaP cement, which was implanted in critical-sized cranial defect in rats and left in place for 2, 4, and 8 weeks. The degradation of the gelatin was monitored using radioiodinated microparticles. After 4 and 8 weeks of implantation, a significantly faster degradation of type A gelatin over type B gelatin was found. Light microscopic analysis of the specimens showed similar bone response concerning implants containing either type A or B gelatin microparticles. At 2 weeks of implantation, a minimal amount of bone formation was observed from the cranial bone toward the implant, while after 8 weeks of implantation an entire layer of newly formed bone was present from the cranial bone toward the implant periphery. Bone ingrowth into the implant was observed at sites of gelatin microparticle degradation, predominantly at the implant periphery. Histomorphometrical evaluation did not reveal significant differences in bone formation between CaP cement incorporated with either type A or B gelatin microparticles during implantation periods up to 8 weeks. In conclusion, this study demonstrates that gelatin type influences the degradation of gelatin microparticles incorporated in CaP cements. However, this difference in degradation and the concomitant subsequent macroporosity did not induce differences in the biological response.

• Effect of platelet-rich plasma on the early bone formation around Ca-P-coated and non-coated oral implants in cortical bone.

  Authors: Dimitris Nikolidakis, Juliette van den Dolder, Joop G C Wolke, John A Jansen

  Clinical oral implants research. 03/2008; 19(2):207-13.

  OBJECTIVES: The purpose of the present study was to investigate the effect of local application of platelet-rich plasma (PRP) on the early healing of cortical bone around Ti implants with twoOBJECTIVES: The purpose of the present study was to investigate the effect of local application of platelet-rich plasma (PRP) on the early healing of cortical bone around Ti implants with two different surface configurations. MATERIAL AND METHODS: Six goats were used in this study. PRP fractions were obtained from a venous blood sample of the goats and administered immediately before implant insertion. PRP was applied via gel preparation and installation of the gel into the implant site, or via dipping of the implants in PRP fraction before insertion. A total of 36 implants (18 non-coated and 18 Ca-P-coated) were placed into the tibial cortical bone. The animals were sacrificed at 6 weeks after implantation and implants with surrounding tissue were prepared for histological examination. Histomorphometrical variables like the percentage of implant surface with direct bone-implant contact and the percentage of new and old bone adjacent to the implant were evaluated. RESULTS: More interfacial bone-to-implant contact was observed for all the three groups of Ca-P-coated implants and the Ti/PRP liquid group. All groups revealed similar percentages of old and new bone adjacent to the implant. CONCLUSIONS: It was concluded that the additional use of PRP did not have any effect on the early cortical bone response to the Ca-P-coated implants, while PRP in a liquid form showed a tendency to increase bone apposition to roughened titanium implants.

• Hard tissue formation in a porous HA/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow.

  Authors: Weibo Zhang, X Frank Walboomers, Gerjo J V M van Osch, Juliette van den Dolder, John A Jansen

  Tissue engineering. Part A. 03/2008; 14(2):285-94.

  The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow,The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic scaffold material, and then either cultured in an osteogenic medium or subcutaneously implanted into nude mice. For cell culture, samples were collected at weeks 0, 1, 3, and 5. Results were analyzed by measuring cell proliferation rate and alkaline phosphatase activity, scanning electron microscopy, and real-time PCR. Samples from the implantation study were retrieved after 5 and 10 weeks and evaluated by histology and real-time PCR. The results indicated that in vitro abundant cell growth and mineralization of extracellular matrix was observed for all types of cells. However, in vivo matured bone formation was found only in the samples seeded with rat bone marrow stromal cells. Real-time PCR suggested that the expression of Runx2 and the expression osteocalcin were important for the differentiation of bone marrow stromal cells, while dentin sialophosphoprotein contributed to the odontogenic differentiation. In conclusion, the limited hard tissue regeneration ability of dental pulp stromal cells questions their practical application for complete tooth regeneration. Repeated cell passaging may explain the reduction of the osteogenic ability of both bone- and dentinal-derived stem cells. Therefore, it is essential to develop new cell culture methods to harvest the desired cell numbers while not obliterating the osteogenic potential.

• Bone response and mechanical strength of rabbit femoral defects filled with injectable CaP cements containing TGF-beta 1 loaded gelatin microparticles.

  Authors: Dennis P Link, Juliette van den Dolder, Jeroen J van den Beucken, Joop G Wolke, Antonios G Mikos, John A Jansen

  Biomaterials. 03/2008; 29(6):675-82.

  This study focused at the potential of transforming growth factor beta 1 (TGF-beta 1) loaded gelatin microparticles to enhance the bone response and mechanical strength of rabbit femoral defectsThis study focused at the potential of transforming growth factor beta 1 (TGF-beta 1) loaded gelatin microparticles to enhance the bone response and mechanical strength of rabbit femoral defects filled with injectable calcium phosphate (CaP)/gelatin microparticle composites. Therefore, TGF-beta1 loaded composites and non-loaded controls were injected in circular defects as created in the femoral condyles of rabbits and were left in place for 4, 8 and 12 weeks. The specimens were evaluated mechanically (push-out test), and morphologically (scanning electron microscopy (SEM), histology, and histomorphometry). The results showed a gradual increase in mechanical strength with increasing implantation periods. Histological and histomorphometrical evaluation showed similar results for both composite formulations regarding histological aspect, new bone formation and bone/implant contact. However, TGF-beta1 loading of the composites demonstrated a significant effect on composite degradation after twelve weeks of implantation. The results of this study showed that CaP/gelatin composites show excellent osteogenic properties and a rapid increase in mechanical strength. The addition of TGF-beta1 significantly enhances the bone remodeling process.

• Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

  Authors: Xuechao Yang, X Frank Walboomers, Juliette van den Dolder, Fang Yang, Zhuan Bian, Mingwen Fan, John A Jansen

  Tissue engineering. Part A. 02/2008; 14(1):71-81.

  Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stemCalcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.

• The response of osteoblast-like cells towards collagen type I coating immobilized by p-nitrophenylchloroformate to titanium.

  Authors: Juliette van den Dolder, John A Jansen

  Journal of biomedical materials research. Part A. 01/2008; 83(3):712-9.

  The scaffold surface composition can be altered by the use of surface coatings. The use of thin coatings will give special surface properties, while the bulk properties of the scaffold are preserved.The scaffold surface composition can be altered by the use of surface coatings. The use of thin coatings will give special surface properties, while the bulk properties of the scaffold are preserved. Collagen type I is known to play an important role during cell adhesion as well as osteoblast differentiation. A common way to coat surfaces is the adsorption method. An alternative way is the use of a protein immobilization method like p-nitrophenyl chloroformate. In this study, we investigated the effect of a collagen type I coating and p-nitrophenyl chloroformate as a protein immobilization method on osteoblast adhesion, proliferation, and differentiation. Titanium fiber meshes were treated with sodium hydroxide (NaOH), followed by p-nitrophenyl chloroformate, and coated with collagen type I. Osteoblast-like cells were seeded into the meshes and cultured for 24 days. The cell attachment, proliferation, and differentiation were measured by using Live and Dead assay, cell counting, DNA analysis, alkaline phosphatase activity assay, calcium content measurement, Real Time PCR (QPCR), and scanning electron microscopy (SEM). Results demonstrated that initially less cells were attached to the covalently bounded collagen meshes (NPC-Col) compared with titanium as control (Ti) and adsorbed collagen meshes (ABS-Col). Further, a decreased growth curve of cells cultured on the NPC-Col meshes was observed in comparison with Ti and ABS-Col meshes. The calcium measurements and SEM pictures revealed that all three surfaces showed differentiation of osteoblast-like cells after 8-24 days. On the basis of our results, we conclude that initially less cells were attached to the NPC-Col meshes and that they had a decreased proliferation rate. Further, we conclude that an adsorbed collagen type I coating stimulated the osteoblastic differentiation of rat bone marrow cells.

• Evaluation of the biocompatibility of calcium phosphate cement/PLGA microparticle composites.

  Authors: Dennis P Link, Juliette van den Dolder, Jeroen J J P van den Beucken, Vincent M Cuijpers, Joop G C Wolke, Antonios G Mikos, John A Jansen

  Journal of biomedical materials research. Part A. 01/2008;

  In this study, the biocompatibility of a calcium phosphate (CaP) cement incorporating poly (D,L-lactic-co-glycolic acid) (PLGA) microparticles was evaluated in a subcutaneous implantation model inIn this study, the biocompatibility of a calcium phosphate (CaP) cement incorporating poly (D,L-lactic-co-glycolic acid) (PLGA) microparticles was evaluated in a subcutaneous implantation model in rats. Short-term biocompatibility was assessed using pure CaP discs and CaP discs incorporating PLGA microparticles (20% w/w) with and without preincubation in water. Long-term biocompatibility was assessed using CaP discs incorporating varying amounts (5, 10, or 20% w/w) and diameter sizes (small, 0-50 mum; medium, 51-100 mum, or large, 101-200 mum) of PLGA microparticles. The short-term biocompatibility results showed a mild tissue response for all implant formulations, irrespective of disc preincubation, during the early implantation periods up to 12 days. Quantitative histological evaluation revealed that the different implant formulations induced the formation of similar fibrous tissue capsules and interfaces. The results concerning long-term biocompatibility showed that all implants were surrounded by a thin connective tissue capsule (<10 layers of fibroblasts). Additionally, no significant differences in capsule and interface scores were observed between the different implant formulations. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth throughout the implants, indicating PLGA degradation and interconnectivity of the pores. The results demonstrate that CaP/PLGA composites evoke a minimal inflammatory response. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth after 12- and 24-weeks indicating PLGA degradation and interconnectivity of the pores. Therefore, CaP/PLGA composites can be regarded as biocompatible biomaterials with potential for bone tissue engineering and advantageous possibilities of the microparticles regarding material porosity. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

• STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

  Authors: Xuechao Yang, Peter M van der Kraan, Juliette van den Dolder, X Frank Walboomers, Zhuan Bian, Mingwen Fan, John A Jansen

  Tissue engineering. 12/2007; 13(11):2803-12.

  Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenicDental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium.

• Pre-culture period of mesenchymal stem cells in osteogenic media influences their in vivo bone forming potential.

  Authors: Harold Castano-Izquierdo, José Alvarez-Barreto, Juliette van den Dolder, John A Jansen, Antonios G Mikos, Vassilios I Sikavitsas

  Journal of biomedical materials research. Part A. 08/2007; 82(1):129-38.

  The objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implantedThe objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implanted in a critical size cranial defect. MSCs were harvested from the bone marrow of 6-8 weeks old male Fisher rats and expanded in vitro in osteogenic media for different time periods (4, 10, and 16 days) in tissue culture plates (TCP), seeded on sintered titanium fiber meshes without the extracellular matrix (ECM) generated in vitro, and implanted in the rat cranium after 12 h. Thirty two adult Fisher rats received the implants, divided in four groups. Three groups were implanted with cells cultured for 4, 10, or 16 days in osteogenic media and at that time their alkaline phosphatase activity and mineral deposition denoted that they were at different stages of their osteoblastic maturation (undifferentiated MSC, committed, and mature Osteoblasts, respectively). MSCs cultured without osteogenic media for 6 days were used as controls. The constructs were retrieved 4 weeks later and processed for histomorphometric analysis. Implants seeded with cells that have been cultured with osteogenic media for only 4 days revealed the highest bone formation. The lowest bone formation was obtained with the implants seeded with MSCs cultured for 16 days in the presence of osteogenic media. The results of this study suggested that the in vitro pre-culture period of MSCs is a critical factor for their ability to regenerate bone when implanted to an orthotopic site.

• Early effect of platelet-rich plasma on bone healing in combination with an osteoconductive material in rat cranial defects.

  Authors: Adelina S Plachokova, Juliette van den Dolder, Paul J Stoelinga, John A Jansen

  Clinical oral implants research. 05/2007; 18(2):244-51.

  OBJECTIVE: The early effect of platelet-rich plasma (PRP) on bone regeneration in combination with dense biphasic hydroxyl apatite (HA)/beta-tricalcium phosphate (TCP) particles (ratio 60%/40%) wasOBJECTIVE: The early effect of platelet-rich plasma (PRP) on bone regeneration in combination with dense biphasic hydroxyl apatite (HA)/beta-tricalcium phosphate (TCP) particles (ratio 60%/40%) was evaluated in rat cranial defects with a diameter of 6.2 mm. We hypothesize that PRP exerts its beneficial effect on bone regeneration within the first and second week after application in a bone defect combined with an osteoconductive material. MATERIALS AND METHODS: Forty-five rats were used in the study, in which always one cranial defect was created. The defects were filled with HA/beta-TCP particles and HA/beta-TCP particles combined with PRP gel. Some defects were also left unfilled as control. One and two weeks after surgery specimens were retrieved for light microscopy [hematoxylin-eosin, trichrome staining (Masson modification Goldner) and basic fuchsin-methylene blue] and micro-CT analysis to evaluate bone formation and neovascularization. One-way analysis of variance was performed on the raw data obtained from micro-CT analyses. RESULTS: The histological evaluation showed no effect of PRP on bone formation and neovascularization for both implantation times. In the first week, the defect closure was evaluated subjectively to be between 10% and 50% in all samples, whereas no difference among the groups appeared to occur. After 2 weeks, complete bridging of the original bone defect was observed for most of the empty defects, as well as for the defects that contained HA/beta-TCP particles. The trichrome staining revealed no difference in the number of blood vessels between the PRP and non-PRP groups for both implantation times. The osteoconductive nature of dense HA/beta-TCP particles was confirmed, as the bone formation was guided by their outer surfaces and resulted in a larger amount of newly formed bone in comparison with the empty defects. The quantitative micro-CT analysis demonstrated a statistically significant difference in new bone formation between the empty defects and defects filled with particles after 1 week of implantation, but there was no difference between the non-PRP and PRP groups. In at the second week, no difference in bone formation among all groups was observed, whereas even the non-filled control defects were almost completely closed. CONCLUSIONS: A 6.2 mm cranial defect is not a critical-sized defect in rats. Rat PRP had no effect on the early stages of bone healing in addition to an osteoconductive material. Dense HA/beta-TCP particles showed a beneficial effect on bone formation already after 1 and 2 weeks of implantation in non-critical-sized cranial defects in rats.

• The cytocompatibility and early osteogenic characteristics of an injectable calcium phosphate cement.

  Authors: Dennis P Link, Juliette van den Dolder, Joop G C Wolke, John A Jansen

  Tissue engineering. 04/2007; 13(3):493-500.

  In this study, the cytocompatibility and early osteogenic characteristics of rat bone marrow cells (RBMCs) on injectable calcium phosphate (CaP) cement (Calcibon) were investigated. In addition toIn this study, the cytocompatibility and early osteogenic characteristics of rat bone marrow cells (RBMCs) on injectable calcium phosphate (CaP) cement (Calcibon) were investigated. In addition to unmodified CaP cement discs, 2 other treatments were given to the discs: preincubation in MilliQ and sintering at different temperatures. After primary culture, RBMCs were dropwise seeded on the discs and cultured for 12 days. The samples were evaluated in terms of cell viability, morphology (live and dead assays and scanning electron microscopy (SEM)), cell proliferation (deoxyribonucleic acid (DNA) analyses), early cell differentiation (alkaline phosphatase (ALP) activity), and physicochemical analyses (x-ray diffraction (XRD)). The live and dead, DNA, and SEM results showed that Calcibon discs without any additional treatment were not supporting osteoblast-like cells in vitro. There were fewer cells, and cell layers were detached from the disc surface. Therefore, different preincubation periods and sintering temperatures were evaluated to improve the cytocompatibility of the CaP cement. Preincubating discs in MilliQ for periods of 1, 4, 8, and 12 weeks resulted in the hydrolysis of alpha-tri calcium phosphate (TCP) into an apatite-like structure with some beta-TCP, as shown with XRD, but the material was not cytocompatible. Sintering the discs between 800 degrees C and 1100 degrees C resulted in conversion of alpha-TCP to beta-TCP with some hydroxyapatite and an increase in crystallinity. Eventually, the discs sintered at 1100 degrees C achieved better cell attachment, more-abundant cell proliferation, and earlier differentiation than other sintered (600 degrees C, 800 degrees C, and 1000 degrees C), preincubated, and unmodified specimens. On basis of our results, we conclude that in vivo results with CaP-based cements do not guarantee in vitro applicability. Furthermore, unmodified Calcibon is not cytocompatible in vitro, although preincubation of the material results in a more-favorable cell response, sintering of the material at 1100 degrees C results in the best osteogenic properties. In contrast to in vivo studies, the Calcibon CaP cement is not suitable as a scaffold for cell-based tissue-engineering strategies.

• Enrichment of osteogenic cell populations from rat bone marrow stroma.

  Authors: Juliette van den Dolder, John A Jansen

  Biomaterials. 02/2007; 28(2):249-55.

  The presence of multiple cell types in bone marrow and their varying proportions from isolation to isolation may count for the considerable variation in the outcome of different experiments. TheThe presence of multiple cell types in bone marrow and their varying proportions from isolation to isolation may count for the considerable variation in the outcome of different experiments. The presence of these multiple subpopulations suggests a need for a method that can purify the osteogenic component, i.e. osteoprogenitors, from other components. The availabilities of monoclonal antibodies recognizing subpopulations of osteoblasts are providing means for antibody-based methods. The cell surface antigens STRO-1, ALP and HOP-26 were used for cell sorting experiments with fluorescence activated cell sorting (FACS). These cell populations were analyzed on differential gene expression, cell proliferation and differentiation into the osteoblastic lineage. The oligo-microarray results showed that only the ALP positive cell population expressed genes of the extracellular matrix; like different collagens, ECM-1 and matrix protease MMP-14. The real-time polymerase chain reaction (QPCR) results showed that STRO-1 and ALP positive cells had an upregulation in expression of lipoprotein lipase, osteocalcin, and collagen type I. Integrin beta-3 was only upregulated for ALP positive cells, while for these cells downregulation occurred for the genes myosine, alkaline phosphatase and integrin beta-1. HOP-26 positive cells showed an upregulation in collagen type I compared to control group. The DNA analysis revealed that the cells of the control group and the HOP-26 positive cells showed a 5 times higher cell growth compared to the STRO-1 and ALP positive cells. The alkaline phosphatase activity showed no activity for the control group. The STRO-1 and ALP positive cells had a higher activity compared to the HOP-26 positive. The calcium measurements revealed only for the control group calcium at day 24. Based on the results of our study, we conclude that the FACS method had no negative effect on the proliferating as well as differentiating response of the cells. Further, we conclude that by using an antibody-based cell selection method, different cell populations with different mRNA expression profiles and different osteogenic characteristics can be obtained.

• Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells.

  Authors: Juliette van den Dolder, Rob Mooren, Annelies P G Vloon, Paul J W Stoelinga, John A Jansen

  Tissue engineering. 12/2006; 12(11):3067-73.

  Platelet-rich plasma (PRP) is a new application of tissue engineering and a developing area for clinicians and researchers. It is a storage vehicle of growth factors (GFs) such as platelet-derivedPlatelet-rich plasma (PRP) is a new application of tissue engineering and a developing area for clinicians and researchers. It is a storage vehicle of growth factors (GFs) such as platelet-derived growth factor (PDGF)- AA, -BB, -AB; transforming growth factor (TGF)-beta1 and -2; platelet-derived epidermal growth factor (PDEGF); platelet-derived angiogenesis factor (PDAF); insulin growth factor-1 (IGF-1); and platelet factor- 4 (PF-4), which are known to influence bone regeneration. However, animal and clinical studies reveal different results with the use of PRP and its effect on bone healing. This could be due to the differences between species, that is, differences between species in GF concentrations or variation in presence of GFs between the various PRPs. In this study, rat bone marrow cells were cultured in PRP-coated wells or in uncoated wells for 16 days in osteogenic medium, and analyzed on cell growth (DNA content) and cell differentiation (alkaline phosphatase [ALP] activity, calcium content, scanning electron microscopy, and QPCR). The concentrations of TGF-beta1, PDGF-AA, PDGF-AB, and PDGF-BB in rat, goat, and human PRP were subsequently determined. The results showed that PRP stimulated initial cell growth and had no effect on ALP activity. The calcium measurements showed a significant increase in calcium at days 8, 12, and 16. The real-time PCR results showed that PRP upregulated osteocalcin at day 1 and collagen type I at day 8. Overall, the immunoassays revealed that human PRP contained higher concentrations of growth factors per platelet compared to rat and goat PRP. Goat PRP showed higher concentrations of growth factors per platelet as compared to rat PRP except for PDGF-BB, which had a higher concentration in rat PRP. TGF-beta1 was the most abundant growth factor in all 3 PRPs. On the basis of our results, we conclude that platelet-rich plasma contains osteo-inductive growth factors, which are probably species related. However, we cannot generalize the results because of large intraspecies variations. Further, we conclude that rat PRP gel stimulates initial growth and differentiation of rat bone marrow cells in vitro.

• Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles.

  Authors: Dennis P Link, Juliette van den Dolder, Wouter J F M Jurgens, Joop G C Wolke, John A Jansen

  Biomaterials. 11/2006; 27(28):4941-7.

  In this study, the mechanical properties of an implanted calcium phosphate (CaP) cement incorporated with 20wt% poly (dl-lactic-co-glycolic acid) (PLGA) microparticles were investigated in a ratIn this study, the mechanical properties of an implanted calcium phosphate (CaP) cement incorporated with 20wt% poly (dl-lactic-co-glycolic acid) (PLGA) microparticles were investigated in a rat cranial defect. After 2, 4 and 8 weeks of implantation, implants were evaluated mechanically (push-out test) and morphologically (Scanning Electron Microscopy (SEM) and histology). The results of the push-out test showed that after 2 weeks the shear strength of the implants was 0.44+/-0.44MPa (average+/-sd), which increased to 1.34+/-1.05MPa at 4 weeks and finally resulted in 2.60+/-2.78MPa at 8 weeks. SEM examination showed a fracture plane at the bone-cement interface at 2 weeks, while the 4- and 8-week specimens created a fracture plane into the CaP/PLGA composites, indicating an increased strength of the bone-cement interface. Histological evaluation revealed that the two weeks implantation period resulted in minimal bone ingrowth, while at 4 weeks of implantation the peripheral PLGA microparticles were degraded and replaced by deposition of newly formed bone. Finally, after 8 weeks of implantation the degradation of the PLGA microparticles was almost completed, which was observed by the bone ingrowth throughout the CaP/PLGA composites. On basis of our results, we conclude that the shear strength of the bone-cement interface increased over time due to bone ingrowth into the CaP/PLGA composites. Although the bone-cement contact could be optimized with an injectable CaP cement to enhance bone ingrowth, still the mechanical properties of the composites after 8 weeks of implantation are insufficient for load-bearing purposes.

• The effect of platelet-rich plasma on the bone healing around calcium phosphate-coated and non-coated oral implants in trabecular bone.

  Authors: Dimitris Nikolidakis, Juliette van den Dolder, Joop G C Wolke, Paul J W Stoelinga, John A Jansen

  Tissue engineering. 10/2006; 12(9):2555-63.

  The effect of local application of autologous platelet-rich plasma (PRP) on bone healing in combination with the use of titanium implants with 2 different surface configurations was investigated. PRPThe effect of local application of autologous platelet-rich plasma (PRP) on bone healing in combination with the use of titanium implants with 2 different surface configurations was investigated. PRP fractions were obtained from venous blood sample of 6 goats and applied via gel preparation and subsequent installation in the implant site or via dipping of the implant in PRP liquid before insertion. Thirty-six implants (18 non-coated and 18 calcium phosphate (CaP) coated) were placed into the goat femoral condyles (trabecular bone). The animals were sacrificed at 6 weeks after implantation, and implants with surrounding tissue were processed for light microscopical evaluation. In addition to subjective description of the histological findings, histomorphometrical variables were also evaluated (the bone-implant contact and the bone mass adjacent to the implant). Significantly more interfacial bone-to-implant contact was observed for all 3 groups of CaP-coated implants and the titanium / liquid group (non-coated implant with PRP liquid) than for the other 2 non-coated titanium groups (with PRP gel or without PRP). The evaluation of the bone mass close to implant surface indicated that all the groups induced a significant increase of the bone mass except the PRP gel groups. On the basis of the observations, it was concluded that magnetron-sputtered CaP coatings can improve the integration of oral implants in trabecular bone. The additional use of PRP did not offer any significant effect on the bone response to the CaP-coated implants, whereas PRP in a liquid form showed a significant effect on bone apposition to roughened titanium implants during the early post-implantation healing phase.

• The bone regenerative effect of platelet-rich plasma in combination with an osteoconductive material in rat cranial defects.

  Authors: Adelina S Plachokova, Juliette van den Dolder, Paul J Stoelinga, John A Jansen

  Clinical oral implants research. 07/2006; 17(3):305-11.

  The effect of platelet-rich plasma (PRP) on bone regeneration, in combination with an osteoconductive material, was evaluated in a rat model. Cranial defects, 6.2 mm in diameter, were filled withThe effect of platelet-rich plasma (PRP) on bone regeneration, in combination with an osteoconductive material, was evaluated in a rat model. Cranial defects, 6.2 mm in diameter, were filled with HA/beta-TCP particles, HA/beta-TCP particles combined with PRP and HA/beta-TCP particles combined with PRP gel, where some were left empty as a control. After 4 weeks of implantation histological, histomorphometrical and micro-computed tomography analyses revealed no difference in new bone formation among the groups. Further, no additional effect of PRP gel in comparison with PRP liquid was detected, except for the increased handling capacity of the graft. These findings suggest that PRP had no positive effect on bone formation in addition to an osteoconductive material after an implantation period of 4 weeks. Also, no negative effect was seen, and neither PRP nor HA/beta-TCP hampered bone ingrowth into the defects.

• Osteoconductive modifications of Ti-implants in a goat defect model: characterization of bone growth with SR muCT and histology.

  Authors: Ricardo Bernhardt, Juliette van den Dolder, Sussane Bierbaum, Rene Beutner, Dieter Scharnweber, John Jansen, Felix Beckmann, Hartmut Worch

  Biomaterials. 07/2005; 26(16):3009-19.

  In this work the osteoconductive potential of coatings for titanium implants using different extracellular matrix components was evaluated. Cylindrical implants with two defined cavities A and B wereIn this work the osteoconductive potential of coatings for titanium implants using different extracellular matrix components was evaluated. Cylindrical implants with two defined cavities A and B were coated with collagen type I, type III, or RGD peptide, and placed in the femur of goats together with an uncoated reference state. Bone contact and volume were determined after 5 and 12 weeks implantation, using both histomorphometry and synchrotron radiation micro computed tomography (SR muCT) as the methods complement each other: SR muCT allows for a high precision of bone detection due to the large number of analysed slices per sample, while histology offers a better lateral resolution and the possibility of additionally determining bone contact. Both methods revealed similar tendencies in bone formation for the differently bio-functionalized implants, with the SR muCT data resulting in significant differences. After 5 and 12 weeks, all three coatings showed a significant increase in bone volume over the uncoated reference, with the highest results for the collagen coatings. The coating consisting of just the RGD-sequence to improve cell adhesion showed only a slight improvement compared with the reference material. For uncoated titanium, RGD, and especially collagen type I, the response in cavity A, situated in denser bone, was stronger than in cavity B. Collagen type III, on the other hand, appeared to be the more effective coating in areas of lesser bone density as represented by cavity B. These results indicate that matrix molecules (or combinations thereof) are capable of generating the appropriate signals for the specific microenvironment around implants and can thus accelerate the bone formation process and increase the stability of implants.

• Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs using a rat cranial critical size defect model.

  Authors: Vassilios I Sikavitsas, Juliette van den Dolder, Gregory N Bancroft, John A Jansen, Antonios G Mikos

  Journal of biomedical materials research. Part A. 01/2004; 67(3):944-51.

  The aim of this study was to investigate the in vivo performance in bone-regenerating capability of cell/scaffold constructs implanted into an orthotopic site. Bone marrow stromal osteoblasts wereThe aim of this study was to investigate the in vivo performance in bone-regenerating capability of cell/scaffold constructs implanted into an orthotopic site. Bone marrow stromal osteoblasts were seeded on titanium fiber mesh scaffolds using a cell suspension (5 x 10(5) cells per scaffold) and cultured for 1, 4, and 8 days under either static or flow perfusion conditions forming six different treatment groups. A total of 16 constructs from each one of the six treatment groups were then implanted into an 8-mm critical size calvarial defect created in the cranium of adult syngeneic male Fisher rats. Half of the constructs from each group were retrieved 7 days postimplantation, and the other half of the constructs were retrieved 30 days postimplantation and examined for new bone formation and tissue response. Constructs retrieved 7 days postimplantation were filled with fibrous tissue and capillaries, but no bone formation was observed in any of the six treatment groups. Constructs retrieved 30 days postimplantation showed bone formation (at least 7 out of 8 constructs in all treatment groups). Titanium fiber meshes seeded with bone marrow stromal osteoblasts and cultured for 1 day under flow perfusion conditions before implantation appeared to give the highest percentage of bone formation per implant (64 +/- 17%). They also showed the highest ratio of critical size cranial defects that resulted in union of the defect 30 days postimplantation (7 out of 8) together with the constructs cultured for 1 day under static conditions before implantation. There were no significant differences between the different treatment groups; this finding is most likely due to the large variability of the results and the small number of animals per group. However, these results show that titanium fiber mesh scaffolds loaded with bone marrow stromal osteoblasts can have osteoinductive properties when implanted in an orthotopic site. They also indicate the importance of the stage of the osteoblastic differentiation and the quality of the in vitro generated extracellular matrix in the observed osteoinductive potential.

• Effect of fibronectin- and collagen I-coated titanium fiber mesh on proliferation and differentiation of osteogenic cells.

  Authors: Juliette van den Dolder, Gregory N Bancroft, Vassilios I Sikavitsas, Paul H M Spauwen, Antonios G Mikos, John A Jansen

  Tissue engineering. 07/2003; 9(3):505-15.

  The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells.The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells. Three main treatment groups were investigated in addition to uncoated titanium fiber meshes: meshes coated with fibronectin, meshes coated with collagen I, and meshes coated first with collagen I and then subsequently with fibronectin. Rat bone marrow cells were cultured for 1, 4, 8, and 16 days in plain and coated titanium fiber meshes. In addition, a portion of each of these coating treatment groups was cultured in the presence of antibodies against fibronectin and collagen I integrins. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, osteocalcin, and calcium content and alkaline phosphatase activity. There were no significant effects of the coatings on cellular proliferation as indicated by the DNA quantification analysis. When antibodies against fibronectin and collagen I integrins were used, a significant reduction (p < 0.05) in cell proliferation was observed for the uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. The different coatings also did not affect the alkaline phosphatase activity of the cells seeded on the coated meshes. However, the presence of antibodies against fibronectin or collagen I integrins resulted in significantly delayed expression of alkaline phosphatase activity for uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. Calcium measurements did not reveal a significant effect of fibronectin or collagen I coating on calcium deposition in the meshes. Also, no difference in calcium content was observed in the uncoated titanium meshes and meshes coated with fibronectin when antibodies against fibronectin or collagen I integrins were present. Meshes coated with both collagen I and fibronectin showed significantly higher calcium content when cultured in the presence of antibodies to collagen and fibronectin integrins. A similar phenomenon was also observed for collagen-coated meshes cultured in the presence of antibodies to fibronectin integrins. No significant differences in osteocalcin content were observed between the treatment groups. However, all groups exposed to antibodies against fibronectin integrins showed a significant decrease in osteocalcin content on day 16. These results show that a fibronectin or collagen I coating does not stimulate the differentiation of rat bone marrow cells seeded in a titanium fiber mesh.

• Bone tissue reconstruction using titanium fiber mesh combined with rat bone marrow stromal cells.

  Authors: Juliette van den Dolder, Edward Farber, Paul H M Spauwen, John A Jansen

  Biomaterials. 06/2003; 24(10):1745-50.

  The study aim was to evaluate the effect of bone marrow stromal cells (BMSCs) cultured in titanium fiber mesh and implanted in a rat cranial defect. A total of 24 titanium meshes were placed in aThe study aim was to evaluate the effect of bone marrow stromal cells (BMSCs) cultured in titanium fiber mesh and implanted in a rat cranial defect. A total of 24 titanium meshes were placed in a tube containing 10 ml BMSC suspension (3 x 10(6)cells/ml) and the tube was rotated on a rotation plate (2 rpm) during 3 h. Thereafter, meshes with cells were subcultured for 1 day under standard conditions. Cell-loaded implants and non-cell-loaded controls were placed in a 8 mm cranial defect and retrieved after 3, 15 and 30 days of implantation. Histology showed that after 3 days of implantation, the mesh porosity of both implant groups was mainly invaded with blood cells. On the other hand, at 15 days of implantation, the cell-loaded implants were filled for 15 +/- 10% of their volume with bone, while the controls showed 1.5 +/- 3.5% of bone. The 30-day cell-loaded implants showed 40 +/- 12.5% of bone and the 30-day control implants 17 +/- 14.5%. At both implantation times the differences were statistically significant. Therefore, we conclude that inoculation of titanium fiber mesh with BMSCs can improve the bone healing capacity of this material.