The aim of the present study was to compare two implant surfaces, the TiOblast (Astra Tech) surface, manufactured by blasting the surface and already present in literature and the Osseospeed (Astra Tech) surface, manufactured by blasting and treating the surface with fluoride ions and recently launched onto the market with the modified surfaces of the latest generation. This study is part of a more extensive research project whose protocol required the insertion of 10 couples of implants; thus in the present discussion partial data are being taken into consideration, with an eye at collecting more data in the future, regarding both microscopy and histomorphometric histological analysis on 5 couples of implants. The purpose of the study is to investigate how the modified surfaces of the latest generation can guarantee a greater osseointegration both from a qualitative and quantitative level compared to the surfaces presently used and that they may represent the first example of “bioactivity”, that is, an active interaction with the processes of new bone formation and tissue healing.
sandblasted surface; fluoride; histology; histomorphometry; microthreads; macrothreads
This study evaluated the initial stability of different implants placed above the bone level in different types of bone.
MATERIALS AND METHODS
As described by Lekholm and Zarb, cortical layers of bovine bone specimens were trimmed to a thickness of 2 mm, 1 mm or totally removed to reproduce bone types II, III, and IV respectively. Three Implant system (Brånemark System® Mk III TiUnite™, Straumann Standard Implant SLA®, and Astra Tech Microthread™-OsseoSpeed™) were tested. Control group implants were placed in level with the bone, while test group implants were placed 1, 2, 3, and 4 mm above the bone level. Initial stability was evaluated by resonance frequency analysis. Data was statistically analyzed by one-way analysis of variance in confidence level of 95%. The effective implant length and the Implant Stability Quotient (ISQ) were compared using simple linear regression analysis.
In the control group, there was a significant difference in the ISQ values of the 3 implants in bone types III and IV (P<.05). The ISQ values of each implant decreased with increased effective implant length in all types of bone. In type II bone, the decrease in ISQ value per 1-mm increase in effective implant length of the Brånemark and Astra implants was less than that of the Straumann implant. In bone types III and IV, this value in the Astra implant was less than that in the other 2 implants.
The initial stability was much affected by the implant design in bone types III, IV and the implant design such as the short pitch interval was beneficial to the initial stability of implants placed above the bone level.
Effective implant length; Initial stability; Implant design; Resonance frequency analysis
Most of the focus in the early dental implant literature is on the bone to titanium interface because a successful Osseo integrated implant requires direct bone contact to the implant surface. The importance of soft tissue in the ability of dental implants to restore function and esthetics has often been underestimated. This paper reviews the pertinent literature on soft tissue healing and management in partially edentulous dental implant patients. Patients seek treatment to replace missing teeth and to improve comfort, function and/or esthetics. Healing around dental implants is affected by the patient’s health, soft and hard tissue contours, and the use and care of the prosthesis, surgical augmentation and placement, and the design of the definitive prosthesis. Several surgical and non-surgical procedures have been proposed to treat the soft tissue deformities in the interproximal areas. This review also discusses the interdental papilla and various approaches to preserve and restore the same. Most of the research was based on scientifically legitimate sources of information obtained from primary literature, other appropriate technical references and searching using various online resources.
Implants; Soft tissue; Surgical management; Non-surgical management
Various bone graft materials have been used for periodontal tissue regeneration. Demineralized freeze-dried bone allograft (DFDBA) is a widely used bone substitute. The current widespread use of DFDBA is based on its potential osteoinductive ability. Due to the lack of verifiable data, the purpose of this study was to assess the osteoinductive activity of different DFDBAs in vitro.
Sarcoma osteogenic (SaOS-2) cells (human osteoblast-like cells) were exposed to 8 mg/mL and 16 mg/mL concentrations of three commercial types of DFDBA: Osseo+, AlloOss, and Cenobone. The effect of these materials on cell proliferation was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The osteoinductive ability was evaluated using alizarin red staining, and the results were confirmed by evaluating osteogenic gene expression using reverse transcription polymerase chain reaction (RT-PCR).
In the SaOS-2 cells, an 8 mg/mL concentration of Osseo+ and Cenobone significantly increased cell proliferation in 48 hours after exposure (P<0.001); however, in these two bone materials, the proliferation of cells was significantly decreased after 48 hours of exposure with a 16 mg/mL concentration (P<0.001). The alizarin red staining results demonstrated that the 16 mg/mL concentration of all three tested DFDBA induced complete morphologic differentiation and mineralized nodule production of the SaOS-2 cells. The RT-PCR results revealed osteopontin gene expression at a 16 mg/mL concentration of all three test groups, but not at an 8 mg/mL concentration.
These commercial types of DFDBA are capable of decreasing proliferation and increasing osteogenic differentiation of the SaOS-2 cell line and have osteoinductive activity in vitro.
Alizarin red; Bone graft; Cell differentiation; Cell proliferation; Regeneration
Multiple biomaterials are clinically available to spine surgeons for performing interbody fusion. Poly-ether-ether-ketone (PEEK) is used frequently for lumbar spine interbody fusion, but alternative materials are also used, including titanium (Ti) alloys. Previously, we showed that osteoblasts exhibit a more differentiated phenotype when grown on machined or grit-blasted titanium aluminum vanadium (Ti6Al4V) alloys with micron-scale roughened surfaces than when grown on smoother Ti6Al4V surfaces or on tissue culture polystyrene (TCPS). We hypothesized that osteoblasts cultured on rough Ti alloy substrates would present a more mature osteoblast phenotype than cells cultured on PEEK, suggesting that textured Ti6Al4V implants may provide a more osteogenic surface for interbody fusion devices.
The aim of the present study was to compare osteoblast response to smooth Ti6Al4V (sTiAlV) and roughened Ti6Al4V (rTiAlV) with their response to PEEK with respect to differentiation and production of factors associated with osteogenesis.
This in vitro study compared the phenotype of human MG63 osteoblast-like cells cultured on PEEK, sTiAlV, or rTiAlV surfaces and their production of bone morphogenetic proteins (BMPs).
Surface properties of PEEK, sTiAlV, and rTiAlV discs were determined. Human MG63 cells were grown on TCPS and the discs. Confluent cultures were harvested, and cell number, alkaline phosphatase–specific activity, and osteocalcin were measured as indicators of osteoblast maturation. Expression of messenger RNA (mRNA) for BMP2 and BMP4 was measured by real-time polymerase chain reaction. Levels of BMP2, BMP4, and BMP7 proteins were also measured in the conditioned media of the cell cultures.
Although roughness measurements for sTiAlV (Sa=0.09±0.01), PEEK (Sa=0.43±0.07), and rTiAlV (Sa= 1.81±0.51) varied, substrates had similar contact angles, indicating comparable wettability. Cell morphology differed depending on the surface. Cells cultured on Ti6Al4V had lower cell number and increased alkaline phosphatase specific activity, osteocalcin, BMP2, BMP4, and BMP7 levels in comparison to PEEK. In particular, roughness significantly increased the mRNA levels of BMP2 and BMP4 and secreted levels of BMP4.
These data demonstrate that rTiAlV substrates increase osteoblast maturation and produce an osteogenic environment that contains BMP2, BMP4, and BMP7. The results show that modifying surface structure is sufficient to create an osteogenic environment without addition of exogenous factors, which may induce better and faster bone during interbody fusion.
Ti6Al4V; PEEK; Osteoblast; BMP; Roughness
The aim of the present clinical study was to determine the local bone density in dental implant recipient sites using computerized tomography (CT) and to investigate the influence of local bone density on implant stability parameters and implant success.
A total of 300 implants were placed in 111 patients between 2003 and 2005. The bone density in each implant recipient site was determined using CT. Insertion torque and resonance frequency analysis were used as implant stability parameters. The peak insertion torque values were recorded with OsseoCare machine. The resonance frequency analysis measurements were performed with Osstell instrument immediately after implant placement, 6, and 12 months later.
Of 300 implants placed, 20 were lost, meaning a survival rate of %. 93.3 after three years (average 3.7 ± 0.7 years). The mean bone density, insertion torque and RFA recordings of all 300 implants were 620 ± 251 HU, 36.1 ± 8 Ncm, and 65.7 ± 9 ISQ at implant placement respectively; which indicated statistically significant correlations between bone density and insertion torque values (p < 0.001), bone density and ISQ values (p < 0.001), and insertion torque and ISQ values (p < 0.001). The mean bone density, insertion torque and RFA values were 645 ± 240 HU, 37.2 ± 7 Ncm, and 67.1 ± 7 ISQ for 280 successful implants at implant placement, while corresponding values were 267 ± 47 HU, 21.8 ± 4 Ncm, and 46.5 ± 4 ISQ for 20 failed implants; which indicated statistically significant differences for each parameter (p < 0.001).
CT is a useful tool to determine the bone density in the implant recipient sites, and the local bone density has a prevailing influence on primary implant stability, which is an important determinant for implant success.
Microarc oxidation (MAO) is a surface treatment that provides nanoporous pits, and thick oxide layers, and incorporates calcium and phosphorus into the coating layer of titanium alloy. We presumed such modification on the surface of titanium alloy by MAO would improve the ability of cementless stems to osseointegrate. We therefore compared the in vitro ability of cells to adhere to MAOed titanium alloy to that of two different types of surface modifications: machined and grit-blasted. We performed energy-dispersive x-ray spectroscopy and scanned electron microscopy investigations to assess the structure and morphology of the surfaces. Biologic and morphologic responses to osteoblast cell lines (SaOS-2) were then examined by measuring cell proliferation, cell differentiation (alkaline phosphatase activity), and αvβ3 integrin. The cell proliferation rate, alkaline phosphatase activity, and cell adhesion in the MAO group increased in comparison to those in the machined and grit-blasted groups. The osteoblast cell lines of the MAO group were also homogeneously spread on the surface, strongly adhered, and well differentiated when compared to the other groups. This method could be a reasonable option for treating the surfaces of titanium alloy for better osseointegration.
Osseointegration is crucial for the long-term success of dental implants and depends on the tissue reaction at the tissue-implant interface. Mechanical properties and biocompatibility make zirconia a suitable material for dental implants, although surface processings are still problematic. The aim of the present study was to compare osteoblast behavior on structured zirconia and titanium surfaces under standardized conditions.
The surface characteristics were determined by scanning electron microscopy (SEM). In primary bovine osteoblasts attachment kinetics, proliferation rate and synthesis of bone-associated proteins were tested on different surfaces.
The results demonstrated that the proliferation rate of cells was significantly higher on zirconia surfaces than on titanium surfaces (p < 0.05; Student's t-test). In contrast, attachment and adhesion strength of the primary cells was significant higher on titanium surfaces (p < 0.05; U test). No significant differences were found in the synthesis of bone-specific proteins. Ultrastructural analysis revealed phenotypic features of osteoblast-like cells on both zirconia and titanium surfaces.
The study demonstrates distinct effects of the surface composition on osteoblasts in culture. Zirconia improves cell proliferation significantly during the first days of culture, but it does not improve attachment and adhesion strength. Both materials do not differ with respect to protein synthesis or ultrastructural appearance of osteoblasts. Zirconium oxide may therefore be a suitable material for dental implants.
To evaluate the feasibility and advantages of constructing a novel tissue engineering bone, using β-tricalcium phosphate (β-TCP) and rat adipose-derived stem cells (ADSCs), modified with BMP2 and BMP7 by lentivirus. In the present study, ADSCs transfected with Lv-BMP2 and Lv-BMP7, alone or together, were seeded on β-TCP scaffold and cultured in vitro. Based on the results of DNA assay, alkaline phosphatase (ALP) activity, alizarin red staining and osteogenic marker genes expression analysis, the BMP2 and BMP7 genes cotransfection group exhibited a higher degree of osteogenic differentiation in vitro. To investigate the in vivo osteogenesis of the tissue engineering bone, the ADSCs/β-TCP constructs were implanted in rat femurs defects for 6 weeks and studied histomorphology and radiography. The results showed that BMP2 and BMP7 genes cotransfection group dramatically enhanced the efficiency of new bone formation than BMP2 group and BMP7 group in vivo. These results demonstrated that it was advantageous to construct tissue engineering bone using ADSCs cotransfected with BMP2 and BMP7 on β-TCP, providing a potential way for treating bone defects.
Titanium dental implants have been used in the treatment of partial or complete edentulism. The height and width of the residual alveolus and surrounding anatomical structures can determine the proper position and path of insertion of dental implants. The following case report describes the treatment of a malpositioned osseo integrated dental implant with an apex perforating the buccal cortex of alveolar bone. A 61-year-old male was referred by his local dentist for the chief complaint of a swelling at site of tooth 14 where an implant was present. Intraoral clinical examination revealed an implant supported porcelain fused to metal crown replacing the maxillary right first premolar. A peri-apical radiograph of the implant revealed no signs of peri-implant bone loss or radiolucency. Surgical exploration and modification of the protruding implant. The area healed uneventfully without the need of explantation of the implant in site of tooth 14. We felt that the conservative treatment provided was prudent and treatment of choice and anticipate that the implant will most likely continue to function for a lifetime.
Dental implant; osseointegration; perforation
Implant related infections are of great concern in modern surgery. In order to improve the implant performance and to reduce implant related infections, titanium (Ti) surface was modified to simultaneously improve cell- materials interactions and antimicrobial activity. Ti surface was first coated with tricalcium phosphate (TCP) using Laser Engineered Net Shaping (LENS™) to improve biocompatibility. Silver (Ag) was then electrodeposited from different concentrations of silver nitrate (AgNO3) solutions to improve the antimicrobial activity. The Ag-TCP coatings were tested for cytotoxicy with human osteoblast cells. The antimicrobial activities of the Ag-TCP coatings were evaluated using Pseudomonas aeruginosa and Staphylococcus aureus bacteria. In vitro bacterial adhesion study indicated a significant reduction in bacterial colony on Ag-TCP coated surfaces when compared to TCP coated surface.
Tricalcium phosphate coating; Silver; cytotoxicity; antimicrobial activity
The study evaluated a null-hypothesis of no differences of fit between stock abutments and CAD/CAM titanium, gold sputtered and zirconia abutments when examined for radiographic adaptation and Scanning Electron Microcopy (SEM) at their inner aspect. The agreement between microscopic and radiographic fit was also assessed.
Implants (Osseospeed, Astra Tech, Mölndal, Sweden) were connected to titanium abutments (Ti-design, Astra Tech, Mölndal, Sweden) (control group n=12), to stock zirconia abutments (Zir-design, Astra Tech) (group 1 n=12) and to third party zirconia abutments (Aadva Zr abutment, GC, Tokyo, Japan) as observed under SEM (JEOL JSM-6060LV, Tokyo, Japan). Two independent operators blindly evaluated the images, according to a three-score scale: perfect adaptation, no complete adaptation, and clear evidence of no adaptation. A Kruskal-Wallis test was applied to assess significant differences in adaptation scores between the groups.
All specimens showed precise SEM adaptation at all tested interfaces and no radiographically apparent gaps. No significant differences were found and therefore the null-hypothesis tested was accepted. Radiographic and SEM scores were in agreement.
CAD/CAM titanium, gold sputtered and zirconia abutments and third-part CAD/CAM zirconia abutments show an adaptation to Astra Tech implants that is comparable to that of stock titanium and zirconia abutments. Clinicians might be able to verify such adaptation with an x-ray. In-vivo studies would be needed to evaluate the clinical outcome of CAD/CAM abutments.
titanium; zirconia; interface; abutment
The aim of this pilot study was to investigate the effect of etched microgrooves on the hydrophilicity of Ti and osteoblast responses.
MATERIAL AND METHODS
Microgrooves were applied on Ti to have 15 and 60 µm width, and 3.5 and 10 µm depth by photolithography, respectively. Further acid etching was applied to create Ti surfaces with etched microgrooves. Both smooth- and acid-etched Ti were used as the controls. The hydrophilicity of Ti was analyzed by determining contact angles. Cell proliferation and osteogenic activity of MC3T3 mouse preosteoblasts were analyzed by bromodeoxyuridine assay and alkaline phosphatase (ALP) activity test, respectively. One-way ANOVA, Pearson's correlation analysis and multiple regression analysis were used for statistics.
Etched microgrooves significantly increased the hydrophilicity of Ti compared to the smooth Ti. 60 µm-wide etched microgrooves significantly enhanced cell proliferation, whereas the osteogenic activity showed statistically non-significant differences between groups. Result of the osteogenic activity significantly correlated with those of hydrophilicity and cell proliferation. Hydrophilicity was determined to be an influential factor on osteogenic activity.
This study indicates that increase in hydrophilicity of Ti caused by etched microgrooves acts as an influential factor on osteogenic activity. However, statistically non-significant increase in the ALP activity suggests further investigation.
Titanium; Etched microgrooves; Hydrophilicity; Osteoblast response
An exogenous supply of growth factors and bioreplaceable scaffolds may help bone regeneration. The aim of this study was to examine the effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells. Rat bone marrow stromal cells were transfected with plasmids encoding mouse TGF-β1 and/or VEGF-A complementary DNAs and cultured for up to 28 days. Furthermore, collagen scaffolds carrying combinations of the plasmids-transfected cells were implanted subcutaneously in rats. The transgenes increased alkaline phosphatase activity, enhanced mineralized nodule formation, and elevated osteogenic gene expressions in vitro. In vivo, messenger RNA expression of osteogenic genes such as BMPs and Runx2 elevated higher by the transgenes. The data indicate that exogenous TGF-β1 and VEGF-A acted synergistically and could induce osteoblastic differentiation of bone marrow stromal cells in both cell culture and an animal model. The results may provide valuable information to optimize protocols for transgene-and-cell-based tissue engineering.
Gene transfer; bone marrow; TGF-β1; VEGF-A; tissue engineering
Many biological polyelectrolytes are capable of undergoing a fluid–fluid phase separation known as complex coacervation. Coacervates were prepared using hyaluronic acid (HA) and a recombinant fusion protein consisting of mussel adhesive motifs and the RGD peptide (fp-151-RGD). The low interfacial energy of the coacervate was exploited to coat titanium (Ti), a metal widely used in implant materials. The coacervate effectively distributed both HA and fp-151-RGD over the Ti surfaces and enhanced osteoblast proliferation. Approximately half of total fp-151-RGD and HA in the solution transferred to the titanium surface within 2 h. Titanium coated with coacervates having high residual negative surface charge showed the highest cell proliferation of preosteoblast cells (MC-3T3) compared to the treatments tested. Indeed, MC-3T3 cells on complex coacervate coated titanium foils exhibited over 5 times greater cell proliferation than bare, HA coated or fp-151-RGD coated titanium.
Complex coacervate; Hyaluronic acid; Mussel adhesive protein; fp-151-RGD; MC-3T3; Biocoating
Functional tissue engineering for bone augmentation requires the appropriate combination of biomaterials, mesenchymal stem cells, and specific differentiation factors. Therefore, we investigated the morphology, attachment, viability, and proliferation of human dental pulp stem cells cultured in xeno-free conditions in human serum medium seeded on β-tricalcium phosphate/poly(l-lactic acid/caprolactone) three-dimensional biomaterial scaffold. Additionally, osteogenic inducers dexamethasone and vitamin D3 were compared to achieve osteogenic differentiation. Dental pulp stem cells cultured in human serum medium maintained their morphology; furthermore, cells attached, remained viable, and increased in cell number within the scaffold. Alkaline phosphatase staining showed the osteogenic potential of dental pulp stem cells under the influence of osteogenic medium containing vitamin D3 or dexamethasone within the scaffolds. Maintenance of dental pulp stem cells for 14 days in osteogenic medium containing vitamin D3 resulted in significant increase in osteogenic markers as shown at mRNA level in comparison to osteogenic medium containing dexamethasone. The results of this study show that osteogenic medium containing vitamin D3 osteo-induced dental pulp stem cells cultured in human serum medium within β-tricalcium phosphate/poly(l-lactic acid/caprolactone) three-dimensional biomaterial, which could be directly translated clinically.
dental pulp stem cells; human serum; β-tricalcium phosphate/poly(l-lactic acid/caprolactone); vitamin D3; osteogenic differentiation
Microstructured and high surface energy titanium substrates increase osseointegration in vivo. In vitro, osteoblast differentiation is increased, but effects of the surface directly on multipotent mesenchymal stem cells (MSCs) and consequences for MSCs in the peri-implant environment are not known. We evaluated responses of human MSCs to substrate surface properties and examined the underlying mechanisms involved. MSCs exhibited osteoblast characteristics (alkaline phosphatase, RUNX2, and osteocalcin) when grown on microstructured Ti; this effect was more robust with increased hydrophilicity. Factors produced by osteoblasts grown on microstructured Ti were sufficient to induce co-cultured MSC differentiation to osteoblasts. Silencing studies showed that this was due to signaling via α2β1 integrins in osteoblasts on the substrate surface and paracrine action of secreted Dkk2. Thus, human MSCs are sensitive to substrate properties that induce osteoblastic differentiation; osteoblasts interact with these surface properties via α2β1 and secrete Dkk2, which acts on distal MSCs.
Many challenges exist in improving early osseointegration, one of the most critical factors in the long-term clinical success of dental implants. Recently, ultraviolet (UV) light-mediated photofunctionalization of titanium as a new potential surface treatment has aroused great interest. This study examines the bioactivity of titanium surfaces treated with UV light of different wavelengths and the underlying associated mechanism. Micro-arc oxidation (MAO) titanium samples were pretreated with UVA light (peak wavelength of 360 nm) or UVC light (peak wavelength of 250 nm) for up to 24 h. UVC treatment promoted the attachment, spread, proliferation and differentiation of MG-63 osteoblast-like cells on the titanium surface, as well as the capacity for apatite formation in simulated body fluid (SBF). These biological influences were not observed after UVA treatment, apart from a weaker effect on apatite formation. The enhanced bioactivity was substantially correlated with the amount of Ti-OH groups, which play an important role in improving the hydrophilicity, along with the removal of hydrocarbons on the titanium surface. Our results showed that both UVA and UVC irradiation altered the chemical properties of the titanium surface without sacrificing its excellent physical characteristics, suggesting that this technology has extensive potential applications and merits further investigation.
Background: The goal of this study was to evaluate the behavior of neonatal rat calvarial osteoblast-like cells cultured on different implant surfaces and exposed once or three times to a 660-nm light-emitting diode (LED).
Methods: An LED with a 660-nm wavelength was applied once or three times to cultured cells on standard and modified sandblasted acid-etched surfaces (SLA and SLActive; Straumann, Basel, Switzerland). To analyze the effect of the LED on cell proliferation, numbers, and viability, cells were cultured on titanium discs, and measurements were taken after 72 h. Cell proliferation rates were assessed using a bromodeoxyuridine immunohistochemical technique. Cell morphologies were evaluated by scanning electron microscopy (SEM).
Results: Osteoblast-like cells proliferated on all tested surfaces, with differences among groups in cell counts and DNA synthesis values. The application of one LED treatment caused a significant increase in cell count in the SLActive group in comparison with the SLA group (p = 0.001), whereas the application of three LED treatments caused a significant decrease in cell count in the SLA group compared with the SLActive group (p < 0.001). After 72 h, the number of cells was highest in the SLActive group exposed once to the LED.
Conclusions: One LED application in the SLActive group resulted in significantly increased cell numbers. However, these findings were not exactly compatible with the SEM findings, which demonstrated fewer cells and weak attachments between cells and to the surface. Thus, further studies using different LED application times are needed to clarify the reason for the increased number of cells that are apparently incapable of attaching to the titanium surfaces after 72 h.
Light-emitting diode; SLA and SLActive surface; primary osteoblast cell culture
Background and methods
Micronanoscale topologies play an important role in implant osteointegration and determine the success of an implant. We investigated the effect of three different implant surface topologies on osteoblast response and bone regeneration. In this study, implants with nanotubes and micropores were used, and implants with flat surfaces were used as the control group.
Our in vitro studies showed that the nanostructured topologies improved the proliferation, differentiation, and development of the osteoblastic phenotype. Histological analysis further revealed that the nanotopology increased cell aggregation at the implant-tissue interfaces and enhanced bone-forming ability. Pushout testing indicated that the nanostructured topology greatly increased the bone-implant interfacial strength within 4 weeks of implantation.
Nanotopography may improve regeneration of bone tissue and shows promise for dental implant applications.
osteoblast; osteointegration; titanium; nanostructure; microstructure
There is increasing application of bone morphogenetic proteins
(BMPs) owing to their role in promoting fracture healing and bone
fusion. However, an optimal delivery system has yet to be identified.
The aims of this study were to synthesise bioactive BMP-2, combine
it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide)
(α-TCP/PLGA) nanocomposite and study its release from the composite.
BMP-2 was synthesised using an Escherichia coli expression
system and purified. In vitro bioactivity was confirmed
using C2C12 cells and an alkaline phosphatase assay. The modified
solution-evaporation method was used to fabricate α-TCP/PLGA
nanocomposite and this was characterised using X-ray diffraction
and scanning electron microscopy. Functionalisation
of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed
and release of BMP-2 was characterised using an enzyme-linked immunosorbent
Alkaline phosphatase activity of C2C12 cells was increased by
the presence of all BMP-2/nanocomposite discs compared with the
presence of a blank disc (p = 0.0022), and increased with increasing
incubation concentrations of BMP-2, showing successful adsorption
and bioactivity of BMP-2. A burst release profile was observed for
BMP-2 from the nanocomposite.
Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction
and was dose-dependent. This material therefore has potential application
as an osteoinductive agent in regenerative medicine.
Osteoinduction; BMP-2; Nanocomposite; Polymer; Ceramics; Tissue engineering; Orthopaedics
In this study, we investigated the in vitro and in vivo biologic activity of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated in 1) a gelatin hydrogel, 2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, 3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and 4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with 125I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivity of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (implant 3, p < 0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than in the gelatin and microsphere/gelatin hydrogels (p < 0.001), however there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.
The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2.
MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days.
At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7.
The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium.
Bone morphogenetic protein-2; Cell differentiation; Cell proliferation; Zirconium oxide
In case of revision or minimal invasive spinal surgery, the amount of autograft possibly harvested from the lamina and the spinous processes is limited. Ekanayake and Shad (Acta Neurochir 152:651–653, 2010) suggest the application of bone shavings harvested via high speed burr additionally or instead, but so far no data regarding their osteogenic potential exist. Aim of the study was to compare the osteogenic potential of bone chips and high speed burr shavings, and to evaluate the applicability of bone shavings as an autograft for spinal fusion. Bone chips and shavings from 14 patients undergoing spinal decompression surgery were analyzed using in vitro tissue culture methods. Osteoblast emigration and proliferation, viability and mineralization were investigated and histological evaluation was performed. Bone chips from all patients showed successful osteoblast emigration after average 5.5 days. In contrast, only 57% of the corresponding bone shavings successfully demonstrated osteoblast emigration within an average time span of 14.8 days. Average osteoblast mobilisation was 1.25 × 106 cells per gram from bone chips and 1.73 × 105 cells per gram from the corresponding bone shavings. No difference was observed regarding cell viability, but population doubling times of bone chip cultures were significantly lower (50.5 vs. 121 h) and mineralization was observed in osteoblasts derived from bone chips only. Although some authors suggest the general applicability of laminectomy bone shavings as autografts for spinal fusion, autologous bone grafts obtained from laminectomy bone chips are superior in terms of cell delivery, cell proliferation and mineralization.
Spinal fusion; Bone graft; Osteoregeneration; Bone chips; High speed burr
As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs).
Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARα and RXRα into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRα or RARα significantly increases trabecular bone and osteoid matrix formation.
Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.