The effect of polyethylenglycol gel on the delivery and osteogenic differentiation of homologous tooth germ-derived stem cells in a porcine model.

OBJECTIVES: The aim of this study was to investigate if bone regeneration can be promoted by homologous transplantation of STRO-1 sorted (STRO-1+) porcine tooth germ mesenchymal stem cells (TGSCs) with the combination of polyethylenglycol (PEG)-based hydrogel and biphasic calcium phosphate (BCP) scaffolds. MATERIAL AND METHODS: TGSCs were isolated from impacted third molars of domestic pigs. Nine critical-sized defects were created as (1) untreated defect; filled with (2) autogenous bone; (3) BCP + PEG; (4) BCP + PEG + unsorted TGSCs; (5) BCP + unsorted TGSCs; (6) BCP + PEG + STRO-1-sorted TGSCs; (7) BCP + STRO-1-sorted TGSCs; (8) BCP + PEG + osteogenic induced unsorted TGSCs; and (9) BCP + PEG + osteogenic induced STRO-1-sorted TGSCs in 20 domestic pigs. CM-DiI labelling was used to track cells in vivo. Histomorphometric assessment of new bone formation was achieved by toluidine blue O staining and microradiography after 1, 2, 4 and 12 weeks posttransplantation. RESULTS: Complete healing was achieved in all defects although defects with PEG hydrogel presented better bone formation while STRO-1+ and unsorted TGSCs showed similar ability to form new bone after 12 weeks. Transplanted cells were seen in defects where PEG hydrogel was used as carriers in contrast to defects treated with cells and only bone grafts. CONCLUSIONS: PEG hydrogel is an efficient carrier for homologous stem cell transplantation. TGSCs are capable of promoting bone healing in critical-sized defects in combination with bone graft and PEG hydrogel. CLINICAL RELEVANCE: This study provides information about the importance of the delivery vehicle for future translational stem cell delivery approaches.

Investigation of structural resorption behavior of biphasic bioceramics with help of gravimetry, µCT, SEM, and XRD.

Resorbable bone substitute materials are widely used for bone augmentation after tumor resection, parallel to implant placement, or in critical size bone defects. In this study, the structural dissolution of a biphasic calcium phosphate bone substitute material with a hydroxyapatite (HA)/tricalcium phosphate (ß-TCP) ratio of 60/40 was investigated by repeatedly placing porous blocks in EDTA solution at 37 °C. At several time points, the blocks were investigated by SEM, µCT, and gravimetry. It was found that always complete 2-3 µm sized grains were removed from the structure and that the ß-TCP is dissolved more rapidly. This selective dissolution of the ß-TCP grains was confirmed by XRD measurements. The blocks were eroded from the outside toward the center. The structure remained mechanically stable because the central part showed a delayed degradation and because the slower dissolving HA grains preserved the integrity of the structure.

Evaluation of a Polyethylene Glycol-Osteogenic Protein-1 System on Alveolar Bone Regeneration in the Mini-Pig.

Alveolar bone regeneration associated with the local release of osteogenic protein-1 (OP-1) from a polyethylene glycol (PEG) scaffold was evaluated in 14 mini-pigs. Following extraction of mandibular teeth and 26-weeks of healing time, standardized bone defects were created bilaterally in the posterior mandibles (3 sites for each hemimandible) that were randomly assigned to treatment groups. Seven treatments groups were compared: 4 different concentrations of the PEG/OP-1 test system (n = 14 for each), a positive control (collagen/OP-1, n = 14), a negative control (PEG only, n = 7) and nontreated defects (n = 7). Each animal provided all test and control groups. The animals were sacrificed after 3 weeks of healing and samples were processed for histology and histomorphometry. Three weeks after implantation, there were positive clinical responses for all test groups. Earlier bone maturation was observed in the test groups that had higher concentrations of OP-1 (0.25, 0.5, or 1 mg/mL) compared to the negative control group (PEG alone), the low concentration group (0.1 mg/mL), and the positive control group (collagen/OP-1). However, histomorphometric quantitative analyses did not reveal any statistical difference between any of the groups. No residual PEG biomaterial or inflammatory responses to the biomaterial or growth factor were observed. This study confirmed the safe local delivery of OP-1 from PEG hydrogel. Alveolar bone regeneration was not statistically different between tests groups, negative control (PEG alone) or commercial positive control (collagen/OP-1). The semi-quantitative analysis, however, showed a trend in favor of the higher concentrations of OP-1 to induce faster bone maturation.

Mandibular reconstruction using a calcium phosphate/polyethylene glycol hydrogel carrier with BMP-2.

AIM: To test the hypothesis that a synthetic hydroxyapatite/ß-tricalcium phosphate (HA/TCP) construct combined with polyethylene glycol (PEG) hydrogel including recombinant human bone morphogenetic proteins-2 (rhBMP-2) enhances new bone formation compared with bone morphogenetic proteins-2 (BMP-2) delivered using the HA/TCP construct alone. MATERIAL AND METHODS: Bilateral mandibular partial thickness 20 × 8 × 8 mm (L × W × H) alveolar defects were surgically created in the edentulated posterior mandible in 18 female minipigs. Randomized into two groups of nine animals each, the alveolar defects either received HA/TCP or HA/TCP/PEG with or without BMP-2 (105 µg/defect) in contra-lateral sites using a split-mouth design. Primary outcome, bone density (%) within four regions of interest, was evaluated following a 4-week healing interval when the animals were killed for histometric analysis. RESULTS: Bone morphogenetic proteins-2 loaded onto HA/TCP constructs significantly enhanced new bone formation compared with HA/TCP controls. Adding PEG apparently obstructed BMP-2 induced bone formation. CONCLUSION: Polyethylene glycol compromises the osteogenic effect of BMP-2.

Early biocompatibility of poly (ethylene glycol) hydrogel barrier materials for guided bone regeneration. An in vitro study using human gingival fibroblasts (HGF-1).

OBJECTIVES: To evaluate the early cellular attachment and viability to modified polyethylene glycol (PEG) hydrogels with the influence of arginine-glycine-aspartic acid (RGD) in an in vitro model system. MATERIAL AND METHODS: Human gingival fibroblasts (HGF-1) were cultured on 6 different modalities of PEG hydrogel in hydrophobic polystyrene wells. A total of 7500 cells/well (10,000 cells/cm(2)) were dispersed over the PEG filled wells and incubated in triplicates for 24 h, 7 and 13 days. Cell numbers were calculated by means of a NucleoCounter. Cell viability was determined by measuring lactate dehydrogenase (LDH). For statistical analysis, nonparametric Kruska-Wallis test followed by Dunetts T3 test were used. RESULTS: All PEG modifications showed good biocompatibility, as demonstrated by low LDH values per cell at the earlier two time points. After 13 days, all PEG modifications showed significantly lower number of cells compared with the controls, and the MX60 configurations demonstrated significantly higher LDH/cell values compared with the other hydrogels. CONCLUSIONS: Modifications of the physio-chemical properties of PEG hydrogels and the addition of RGD and spacers influenced the initial cellular response of cultured HGF-1 cells. With the exception of MX60 after 13 days, all PEG formulations performed similarly well. Early cellular response should be considered when developing PEG-based material for clinical purposes.

Biodegradation and bone formation of various polyethylene glycol hydrogels in acute and chronic sites in mini-pigs.

OBJECTIVE: i) To test whether or not pH modifications of a PEG hydrogel matrix influence degradation time and bone regeneration in acute and unprepared (chronic) defects; and ii) to test whether or not the addition of a PEG hydrogel to hydroxyapatite/tricalciumphosphate (HA/TCP) can further enhance bone regeneration compared to HA/TCP alone in acute defects. MATERIALS AND METHODS: In 11 mini-pigs, three acute standardized defects and one chronic site were prepared in each hemi-mandible. The following treatment modalities were applied in acute defects: PEG hydrogel regular (PEG 8.7), PEG hydrogel pH-modified plus (PEG 9.0), PEG hydrogel pH-modified minus (PEG 8.4), PEG 8.7 mixed with HA/TCP granules (PEG-HA/TCP), HA/TCP granules (HA/TCP), and empty control (control). In chronic sites, PEG 8.7 and PEG 9.0 were applied. Subsequently primary wound closure was obtained and animals sacrificed at 10 (n = 6) and 21 days (n = 5). Descriptive histology and histomorphometric analyses were performed including measurements for newly formed bone, remaining hydrogel, and percent defect fill. Standard descriptive statistics were calculated, and regression analysis used to determine the difference between treatments, taking into account relevant factors and correction for multiple comparisons. RESULTS: In acute defects, the amount of newly formed bone increased statistically significantly over time for all treatments. The increase was higher for PEG 8.7 (35.9%) compared with PEG 8.4 and PEG 9.0 and was higher for PEG-HA/TCP (24.7%) than for HA/TCP (14.6%). The remaining hydrogel ranged between 7.6 ± 13.3% for PEG 8.4 and 17.7 ± 12.8% for PEG 8.7 at 10 days. At 21 days, no remaining hydrogel was found except for PEG-HA/TCP (11.5 ± 10.4%). In chronic sites, at 10 days, the remaining hydrogel covered 29.5 ± 10.3% (PEG 9.0) and 25.6 ± 21.8% (PEG 8.7) of the area. At 21 days, the amount of hydrogel (29.7 ± 31.7% for PEG 9.0; 1.4 ± 2.5% for PEG 8.7) decreased, while the amount of bone increased to 14.0 ± 16.3% for PEG 9.0 and to 37.9 ± 15.7% for PEG 8.7. CONCLUSIONS: The PEG hydrogel matrix with a mid-range pH (PEG 8.7) may serve as a matrix for localized bone regeneration with or without the addition of a bone substitute material. This was demonstrated by enhanced bone regeneration in acute and chronic defects compared with control hydrogels and HA/TCP alone.

Critical size defect regeneration using PEG-mediated BMP-2 gene delivery and the use of cell occlusive barrier membranes - the osteopromotive principle revisited.

OBJECTIVE: The objective of this study was to investigate if osseous regeneration can be accelerated by involvement of periosteal tissue. Bone defect regeneration could be accelerated by the involvement of periosteal tissue if osteogenic cell signalling is maintained within the defect. It was questioned if local cell-mediated BMP-2 gene delivery makes a cell occlusive membrane dispensable during bone critical size defect regeneration. METHODS: PEG matrix (degradation time 10 days) and PEG membrane (degradation time 120 days) were used in the pig calvarial model. Cylindrical (1 × 1 cm) critical size defects (CSD) (9 per animal; 20 animals) were filled with: (i) particulated autologous bone, covered with PEG membrane (group 1); (ii) HA/TCP, covered with PEG membrane (group 2); (iii) HA/TCP, mixed with PEG matrix (group 3); and (iv) HA/TCP mixed with BMP-2-transfected osteoblasts and PEG matrix (group 4). BMP-2/4 gene transfer: liposomal in vitro transfection of BMP-2/V5-tag fusion-protein. Quantitative histomorphometry (toluidine blue staining) after 2, 4 and 12 weeks assessed bone formation. Semiquantitative immunohistochemistry estimated the expression of BMP-2, V5-tag, Runx-2 and Sox9. RESULTS: PEG matrix embedded BMP-2 expressing cells presented higher bone formation (P < 0.05) than HA/TCP + PEG matrix defect filling or PEG membrane covering (HA/TCP filling) after 12 weeks. Highest expression of BMP-2, Runx-2 and lowest expression of fibrous tissue marker Sox9 was seen in the BMP-2 group. CONCLUSION: PEG matrix embedded BMP-2 expressing cells are capable to maintain osteogenic signalling and to accelerate osseous defect regeneration in absence of a cell occlusive membrane.

PEG matrix enables cell-mediated local BMP-2 gene delivery and increased bone formation in a porcine critical size defect model of craniofacial bone regeneration.

PURPOSE: This study addressed the suitability of a polyethylene glycol (PEG) matrix as scaffold for cell-mediated local BMP-2 gene transfer in a calvarial critical size defect (CSD) model. MATERIALS AND METHODS: PEG matrix (degradation time 10 days) and PEG membrane (degradation time 120 days) were used in the pig calvarial model. Cylindrical (1 × 1 cm) CSD (9 per animal; 20 animals) were filled with: (i) HA/TCP, covered by PEG membrane (group 1); (ii) HA/TCP, mixed with PEG matrix (group 2); and (iii) HA/TCP mixed with BMP-2 transfected osteoblasts and PEG matrix (group 3). BMP-2/4 gene transfer: liposomal in vitro transfection of BMP-2/V5-tag fusion-protein. Quantitative histomorphometry (toluidine blue staining) after 2, 4 and 12 weeks assessed bone formation. Semiquantitative immunohistochemistry estimated the expression of BMP-2 and V5-tag. RESULTS: Group 3 showed significantly higher new bone formation than groups 1, 2 at 4 (P < 0.05) and 12 (P < 0.02) weeks. BMP-2-V5-tag was detected for 4 weeks. BMP-2 expression in group 3 was higher compared to all other groups after 2 and 4 (P < 0.02) weeks. CONCLUSIONS: The PEG matrix serves as scaffold for cell-mediated BMP-2 gene delivery in guided bone regeneration facilitating cell survival and protein synthesis for at least 4 weeks. Local BMP-2 gene delivery by PEG matrix-embedded cells leads to increased bone formation during critical size defect regeneration.

The effect of enamel matrix proteins on the spreading, proliferation and differentiation of osteoblasts cultured on titanium surfaces.

Modifications of implant surface topography and chemistry have proven a means to enhance osseointegration, a process that ensures the stability of bone-contacting devices, including titanium dental implants. The commercial product Emdogain is an enamel matrix derivative (EMD) extracted from porcine teeth commonly used in periodontal surgery, where it has been shown to potentiate regeneration of bone. The aim of the present study was to evaluate the effect of EMD on the attachment, proliferation and differentiation of osteoblasts on titanium surfaces in vitro. Pickled (smooth) and SLA (roughened) titanium discs were coated with EMD or left uncoated. Primary rat calvarial osteoblasts were cultured on each surface from 1h to 4 weeks. EMD significantly increased cell spreading and proliferation at time points ranging from 3 to 7 days on both topographies. Alkaline phosphatase activity was significantly increased on EMD-coated titanium compared with titanium alone. Moreover, there was a 6 fold increase in levels of mRNA encoding bone sialoprotein and osteocalcin in osteoblasts cultured on EMD-coated titanium surfaces compared with uncoated surfaces. We conclude that coating of titanium with EMD enhances the proliferation and differentiation of osteoblasts irrespective of the titanium substratum topography.

Biofilm on dental implants: a review of the literature.

PURPOSE: The aim of this article was to review the current literature with regard to biofilm formation on dental implants and the influence of surface characteristics (chemistry, surface free energy, and roughness) of dental implant and abutment materials and their design features on biofilm formation and its sequelae. MATERIALS AND METHODS: An electronic MEDLINE literature search was conducted of studies published between 1966 and June 2007. The following search terms were used: biofilm and dental implants, biofilm formation/plaque bacterial adhesion and implants, plaque/biofilm and surface characteristics/roughness/surface free energy of titanium dental implants, implant-abutment interface and plaque/biofilm, biofilm and supragingival/subgingival plaque microbiology, biofilm/plaque and implant infection, antibacterial/bacteriostatic titanium, titanium nanocoating/nanopatterning, antimicrobial drug/titanium implant. Both in vitro and in vivo studies were included in this review. RESULTS: Fifty-three articles were identified in this review process. The articles were categorized with respect to their context on biofilm formation on teeth and dental implant surfaces and with regard to the influence of surface characteristics of implant biomaterials (especially titanium) and design features of implant and abutment components on biofilm formation. The current state of literature is more descriptive, rather than providing strong data that could be analyzed through meta-analysis. Basic research articles on surface modification of titanium were also included in the review to analyze the applications of such studies on the fabrication of implant surfaces that could possibly decrease early bacterial colonization and biofilm formation. CONCLUSIONS: Increase in surface roughness and surface free energy facilitates biofilm formation on dental implant and abutment surfaces, although this conclusion is derived from largely descriptive literature. Surface chemistry and the design features of the implant-abutment configuration also play a significant role in biofilm formation.

Biodegradation of different synthetic hydrogels made of polyethylene glycol hydrogel/RGD-peptide modifications: an immunohistochemical study in rats.

AIM: The aim of the present study was to investigate the pattern of biodegradation of different polyethylene glycol (PEG) hydrogel/RGD-peptide modifications in rats. MATERIAL AND METHODS: Two different hydrogels were employed: (i) a combination of four-arm PEG-thiol, M(n)=2.3 kDa, and eight-arm PEG-acrylate, M(n)=2.3 kDa (PEG1); and (ii) a combination of four-arm PEG-thiol, M(n)=2.3 kDa, and four-arm PEG-acrylate, M(n)=15 kDa (PEG2). Both PEG1 and PEG2 were either used alone or combined with a nine amino acid cys-RGD peptide (RGD). A non-cross-linked porcine type I and III collagen membrane [BioGide (BG)] served as control. Specimens were randomly allocated in unconnected subcutaneous pouches separated surgically on the back of 60 wistar rats, which were divided into six groups (1, 2, 4, 8, 16, and 24 weeks). Specimens were prepared for histological (tissue integration, foreign body reactions, biodegradation) and immunohistochemical (angiogenesis) analysis. RESULTS: All materials investigated revealed unimpeded and comparable tissue integration without any signs of foreign body reactions. While BG exhibited transmembraneous blood vessel formation at 1 week, all PEG specimens were just surrounded by a well-vascularized connective tissue. The hydrolytic disruption of PEG1 and PEG1/RGD specimens was associated with an ingrowth of blood vessels at 4 weeks. Biodegradation times were highest for PEG1 (24 weeks)>PEG1/RGD (16 weeks)>BG (4 weeks)>PEG2=PEG2/RGD (2 weeks). CONCLUSION: Within the limits of the present study, it was concluded that (i) all materials investigated revealed a high biocompatibility and tissue integration, and (ii) hydrogel biodegradation was dependent on PEG composition.

A novel, tissue occlusive poly(ethylene glycol) hydrogel material.

The use of guided bone regeneration (GBR) techniques requires new materials meeting the needs of clinical application. Design criteria for GBR devices are biocompatibility, tissue occlusion, space provision, and clinical manageability. This study evaluates a novel biodegradable poly (ethylene glycol) (PEG) based material as tissue occlusive membrane. A subcutaneous implant model in rats was developed to test the barrier function of the PEG hydrogels over time. Fourteen rats received three membrane implants and two positive controls each. Explants were collected over a period of 7 months. Histological analysis revealed that for at least 4 months cellular infiltration in the membrane explants was lower than 1% of that of the positive controls. Therefore, the PEG based hydrogel can be regarded as tissue occlusive during this period of time. A barrier function seems to be maintained for up to 6 months. In vitro degradation studies performed with the same PEG constructs confirm the in vivo result. In conclusion, our results indicate that this novel PEG-based material has potential for use as a GBR barrier membrane.

Evaluation of an in situ formed synthetic hydrogel as a biodegradable membrane for guided bone regeneration.

The aim of the present study was to test whether or not the application of an in situ formed synthetic hydrogel made of polyethylene glycol (PEG) used as a biodegradable membrane for guided bone regeneration will result in the same amount of bone regeneration as with the use of an expanded polytetrafluoro-ethylene (ePTFE) membrane. In eight New Zealand White rabbits, four evenly distributed 6 mm diameter defects were drilled into the calvarial bone. Three treatment modalities were evenly distributed among the 32 defects: hydroxyapatite (HA)/tricalciumphosphate (TCP) granules covered at the outer and inner surface with a PEG membrane (test), HA/TCP granules covered at the outer and inner surface with an ePTFE membrane (positive control) and HA/TCP granules alone without membranes (negative control). After 4 weeks, the animals were sacrificed and the calvarial bones were removed. The area fraction of newly formed bone was determined by histomorphometrical analysis of the vertical sections from the middle of the defect and by micro-computed tomography of the entire defect. Multiple regression analysis (SAS GLM) was used to model the amount of new bone formation. The quantitative histomorphometric analysis clearly revealed higher values of newly formed bone for the two membrane groups compared with the negative control group. The average area fractions of newly formed bone measured within the former defect amounted to 20.3+/-9.5% for the PEG membrane, 18.9+/-9.9% for the ePTFE membrane, and 7.3+/-5.3% for the sites with no membrane. The micro-computed tomography also showed higher values of new bone formation for the PEG and for the ePTFE groups compared with the negative control group. The GLM revealed a highly significant effect of the treatment on the amount of bone formation (P=0.0048). The values for the negative control group were significantly lower than the ones found in the PEG membrane group (P=0.0017), whereas the ePTFE membrane group showed no significant difference from the PEG membrane group. It is concluded that the PEG membrane can be used successfully as a biodegradable barrier membrane in the treatment of non-critical-size defects in the rabbit skull, and leads to similar amounts of bone regeneration as an ePTFE membrane.