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Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy.

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. 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.

Here, we report the isolation of progenitor cells from pig skeletal muscle tissue fragments. Muscle progenitor cells were stimulated to migrate from protease-digested tissue fragments and cultured in the presence of 5 ng/ml basic fibroblast growth factor. The cells showed a sustained long-term expansion capacity (>120 population doublings) while maintaining a normal karyotype. The proliferating progenitor cells expressed PAX3, DESMIN, SMOOTH MUSCLE ACTIN, VIMENTIN, CD31, NANOG and THY-1, while MYF5 and OCT3/4 were only expressed in the lower or higher cell passages. Myogenic differentiation of porcine progenitor cells was induced in a coculture system with murine C2C12 myoblasts resulting in the formation of myotubes. Further, the cells showed adipogenic and osteogenic lineage commitment when exposed to specific differentiation conditions. These observations were determined by Von Kossa and Oil-Red-O staining and confirmed by quantitative RT-PCR analysis. In conclusion, the porcine muscle-derived progenitor cells possess long-term expansion capacity and a multilineage differentiation capacity.

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 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.)

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 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.

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.

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 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.

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 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 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., (Copyright 2007 Wiley Periodicals, Inc.)

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 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.

The aim of this study was to examine the bone augmentation properties of an injectable composite consisting of PLGA microspheres/CaP cement (20/80), and the additional effect of loading PLGA microspheres with TGF-beta1 (200 ng). For this purpose, PLGA/CaP composites (control) and PLGA/CaP composites loaded with TGF-beta1 (test group) were injected on top of the skulls of 24 Wistar rats. Each rat received 2 materials from the same experimental group, and in total 48 implants were placed (n = 8). After 2, 4, and 8 weeks the results were evaluated histologically and histomorphometrically. The contact length between the implants and newly formed bone increased in time, and was significantly higher for the TGF-beta1-loaded composites after 2 weeks. Also, bone formation was significantly higher for the TGF-beta1-loaded composites (18.5% +/- 3) compared to controls (7.21% +/- 5) after 8 weeks of implantation. Immunohistochemical staining demonstrated massive inflammatory infiltrates in both groups, particularly at 2 weeks, which decreased substantially at 4 and 8 weeks. In conclusion, injectable PLGA/CaP composites stimulated bone augmentation in a rat model. The addition of TGF-beta1 to the composite significantly increased bone contact at 2 weeks and enhanced new bone formation at 8 weeks.

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 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., (Copyright 2007 Wiley Periodicals, Inc.)

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%) 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 aim of the current study was to determine whether STRO-1 selection is an effective approach for purifying rat dental pulp stem cells, and especially whether such selection is beneficial on the multilineage differentiation capacity, i.e. whether selection will account for a higher rate of differentiation or lesser variability. In this study, two cell populations (STRO-1(+) and non-sorted cells) were cultured under conditions promoting neurogenic, adipogenic, myogenic and chondrogenic differentiation. Results of light microscopy, histochemistry, and immunohistochemistry showed that STRO-1(+) cells were capable of advancing into all four differentiation pathways under the influence of inductive media. Quantitative PCR and statistical analysis on specific differentiation markers confirmed that there were significant upregulations in STRO-1(+) cells compared to the other populations, during induction culture. On the basis of our results, we concluded that: (a) rat STRO-1(+) dental pulp stem cells are capable of differentiating towards multilineage cell types, including neural cells, adipocytes, myocytes and chondrocytes; (b) the STRO-1(+) population has a more defined multilineage potential compared to non-sorted cells, probably because of its more homogeneous nature. ., (2007 John Wiley & Sons, Ltd)

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 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.

The presence of heterogeneous cell populations in dental pulp may count for the considerable variation in the outcome of in vitro and in vivo experiments. Here, we intended to determine whether a minor cell sub-population of high proliferation and odontogenic potential existed among a larger compartment of perhaps more committed progenitors. In this study, the STRO-1 antigen, defining a mesenchymal stem cell or progenitor subpopulation, was used for separating rat dental pulp cells with fluorescence-activated cell sorting (FACS). Subsequently, the STRO-1 positive cells were tested for their ability to differentiate towards an odontoblast-like phenotype. Three cell populations (STRO-1 positive, STRO-1 negative, and non-sorted cells) were cultured in odontogenic medium containing dexamethasone and beta-glycerophosphate. Cultures were analyzed by light- and scanning electron microscopy (SEM), and assessed for proliferation, ALP activity, and calcium content. Results showed that the STRO-1 positive cell population was able to differentiate into the odontoblast phenotype, similar to the non-sorted population. The negative cells however showed a fibroblast-like phenotype. SEM and real-time PCR confirmed such results. In conclusion, the STRO-1 selection proved applicable for rat-derived material, to obtain a cell population which is more homogeneous. This positive cell fraction was capable of differentiating into the odontogenic pathway, whereas the negative fraction was not. However, the effect was not always advantageous, when compared to non-sorted cells., (2007 John Wiley & Sons, Ltd)

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. 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 (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.

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 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 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 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.

Little is known about the ability of peptide-coated surfaces to influence cell responses in vivo. Many studies have demonstrated that peptide-modified surfaces influence cell responses in vitro. Integrins, which bind specifically short peptide sequences, are responsible for these cell responses. In this way, information can be transmitted to the nucleus through several cytoplasmic signaling pathways. The peptide sequence Arg-Gly-Asp (RGD peptide) plays an important role in osteoblast adhesion. The present study was designed to investigate new bone formation in a porous titanium (Ti) fiber mesh implant, which was coated with cyclic RGD peptide. The RGD-Ti implants were inserted into the cranium of a rabbit and were compared with porous titanium fiber mesh disks without RGD sequence (Ti) and with an open control defect. Histologic and histomorphometric examinations were performed 2, 4, and 8 weeks postoperatively. A significant increase in bone formation, or bone ingrowth, was seen in the RGD-Ti group compared with the Ti group after 4 and 8 weeks. All control defects stayed open in all three periods. It was concluded that the use of cyclic RGD peptide in combination with titanium fiber mesh has a positive effect on bone formation in vivo in a rabbit animal model.

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 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.

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 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., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 944-951, 2003)

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.

The objective of this study was to examine the osteoinductive capacity of different concentrations of BMP-2 on bone marrow stromal cells in vitro. Further, we intended to determine whether titanium provided with an increased surface roughness is more efficient in osteoblast differentiation than machined titanium. Therefore, 20,000 cells/ml were seeded and cultured on machined and grit-blasted titanium discs for 4, 8 and 16 days. Different concentrations of rhBMP-2 (0, 10, 100, 1000 ng/ml) were supplemented to the medium for 8 days of culturing. To evaluate cellular proliferation and differentiation, specimens were examined for DNA, alkaline phosphatase activity, and calcium content. Morphological appearance of the specimens at 8 and 16 days of incubation was evaluated using scanning electron microscopy. Two separate experimental runs were performed. Evaluation of the DNA and alkaline phosphatase data revealed that a significant difference existed for these data between both experimental runs. Further analysis of the DNA figures learned that roughening of the titanium surface and addition of BMP-2 had no effect on cell proliferation. The alkaline phosphatase analysis and calcium measurements revealed that BMP-2 stimulated the early differentiation of osteogenic cells on machined titanium substrates in a dose-dependent manner. After 16 days of culture, no significant differences in calcium content could be observed anymore between machined and roughened titanium surfaces. Further, the data revealed that the machined surfaces showed a significant increase in calcium deposition when 100 and 1000 ng/ml BMP-2 were supplemented to the medium. However, the roughened surfaces showed this significant enhancement in calcium content only with 1000 ng/ml BMP-2. In addition, SEM evaluation revealed a dose-dependent response to BMP-2. Increasing BMP-2 concentrations resulted in more calcified globular accretions on bone surfaces than when no BMP-2 was added. On the basis of our results, we conclude that (1) due to the heterogeneous nature of bone marrow, experimental results with primary rat bone marrow cells are difficult to reproduce from one experiment to the other, and (2) addition of rhBMP-2 in the medium stimulates the early differentiation and matrix mineralization of osteogenic cells on machined titanium surfaces in a dose-responsive manner. Further, we concluded that our roughened titanium surfaces had no effect on proliferation and differentiation of primary derived rate bone marrow cells.

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 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., (Copyright 2003 Elsevier Science Ltd.)

The objective of the present study was to learn more about the effect of seeding and loading techniques on the osteogenic differentiation in vitro of rat bone marrow cells into titanium fiber mesh. This material was used as received or subjected to glow discharge treatment (RFGD). The seeding methods that were used included a so-called droplet, cell suspension (high and low cell density), and rotating plate method. Osteogenic cells were cultured for 4, 8, and 16 days into titanium fiber mesh. DNA, osteocalcin, scanning electron microscopy (SEM) analysis, and calcium measurements were used to determine cellular proliferation and differentiation. DNA analysis of the differently seeded specimens showed that proliferation proceeded faster in the first versus second run for droplet and cell suspension samples. No clear and distinct additional effect was found when RFGD treatment was used. Statistical analyses revealed that high cell density and low rotational speed resulted always in a significantly higher DNA content. Calcium measurements and osteocalcin analysis showed that using high cell densities during inoculation of the scaffolds prevented the occurrence of differences between experimental runs. SEM examination showed that for droplet and cell suspension samples cells were present at only one side of the mesh. The mesh side where the cell sheet was observed depended on the additional use of glow discharge treatment. On these materials, the cells had penetrated through the meshes and formed a cell sheet at the bottom side. When rotation was used, no cell sheet was formed and cells had invaded the meshes and were growing around the titanium fibers. On the basis of our results, we conclude that (1). titanium fiber mesh is indeed suitable to support the osteogenic expression of bone marrow cells, and (2). changing the initial cell density as well as the use of dynamic seeding methods can influence the osteogenic capacity of the scaffold.

The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system. At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh., (Copyright 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 235-241, 2003)

The objective of this study was to examine the effect of cell culture time on bone formation by rat bone marrow cells seeded in titanium fiber mesh. As a seeding technique, a high cell suspension was used (3 x 10(6) cells/mL). Therefore, 30 meshes were repeatedly rotated in a 10 mL tube (containing 30 x 10(6) cells) on a rotation plate (2 rpm) for 3 h. Osteogenic cells were cultured for 1, 4, and 8 days on titanium fiber mesh and finally implanted subcutaneously in rats. Meshes without cells were also implanted subcutaneously in rats. DNA and scanning electron microscopy (SEM) analyses and calcium measurements determined cellular proliferation and differentiation during the in vitro incubation period of the mesh implants. Four weeks after implant insertion, the animals were sacrificed. The implants, with their surrounding tissue, were retrieved and prepared for histologic evaluation and calcium measurements. DNA analysis of the in vitro experiment showed a lag phase from day 1 through day 4, but a 42% increase in DNA between days 4 and 8. SEM and calcium measurements indicated an increase in calcium from day 1 to day 4, yet only a small but significant increase from days 4 to 8. Histologic analysis demonstrated that bone was formed in all day 1 and day 4 implants, and that the bone-like tissue was present uniformly through the meshes. The bony tissue was morphologically characterized by osteocytes embedded in a mineralized matrix, with a layer of osteoid and osteoblasts at the surface. The day 8 implants showed only calcium phosphate deposition in the titanium fiber mesh. Calcium measurements of the implants revealed that calcification in day 1 implants was significantly higher (p < 0.05) compared to day 4 and day 8 implants. No significant difference in calcium content existed between day 4 and day 8 implants. On the basis of our results, we conclude that 1) bone formation was generated more effectively in osteogenic cells by a short culture time after seeding in titanium fiber mesh; 2) dynamic cell seeding is probably more effective than static cell seeding; and 3) selection of the right cells from the heterogenous bone marrow population remains a problem., (Copyright 2002 Wiley Periodicals, Inc.)

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

The gram-positive eubacterium Bacillus subtilis is the organism with the largest number of paralogous type I signal peptidases (SPases) known. These are specified both by chromosomal and plasmid-borne genes. The chromosomally encoded SPases SipS and SipT have a major function in precursor processing, and cells depleted of SipS and SipT stop growing and die. In this study, we show that the SPase SipP, specified by the B. subtilis plasmid pTA1015, can functionally replace SipS and SipT, unlike the three chromosomally encoded SPases with a minor function in protein secretion (i.e., SipU, SipV, and SipW). Unexpectedly, SipP is not specifically required for the processing and secretion of Orf1p, which is specified by a gene that is cotranscribed with sipP. These two genes form a conserved structural module of rolling-circle plasmids from B. subtilis. As previously shown for the chromosomal sipS and sipT genes, the transcription of plasmid-borne copies of sipP is temporally controlled, reaching maximal levels during the post-exponential growth phase when the cells secrete proteins at high levels. However, increased transcription of sipP starts at the end of exponential growth, about 2 h earlier than that of sipS and sipT. These data suggest that SipP fulfills a general role in the secretory precursor processing of pTA1015-containing cells.