Knut N. Leknes

Alveolar ridge augmentation using implants coated with recombinant human bone morphogenetic protein-2: radiographic observations.

OBJECTIVES Effective carrier technologies and dosing appear critical for the successful use of bone morphogenetic proteins (BMPs). This study evaluated radiographically the potential of a purpose-designed titanium porous-oxide implant surface combined with recombinant human BMP-2 (rhBMP-2) to stimulate alveolar ridge augmentation. MATERIAL AND METHODS Twelve young-adult Labrador dogs were used. Three 10-mm titanium implants per jaw quadrant were placed 5 mm into the alveolar ridge following extraction of the premolar teeth and reduction of alveolar ridge. Six animals received implants coated with rhBMP-2 at 0.75 or 1.5 mg/ml randomized to contralateral jaw quadrants. Another six animals received implants coated with rhBMP-2 at 3 mg/ml or uncoated control using the same split-mouth design. The mucoperiosteal flaps were advanced, adapted, and sutured to submerge the implants. Radiographic registrations were made immediately postsurgery (baseline), and at weeks 4 and 8 (end of study). RESULTS rhBMP-2-coated implants exhibited robust radiographic bone formation extending to and above the implant platform from week 4 (P<0.01). Some rhBMP-2-coated implants showed voids within the newly formed bone that gradually resolved and/or implant displacement, being severe in two animals receiving implants coated with rhBMP-2 at 3 mg/ml. Controls showed limited, if any, new bone formation at weeks 4 and 8 postsurgery. There were no significant differences among the rhBMP-2 groups in bone gain. CONCLUSIONS The titanium porous-oxide surface serves as an effective carrier for rhBMP-2, showing a clinically significant potential to stimulate local bone formation. With the carrier technology used, therapeutic dosage appears to be in the range of 0.75-1.5 mg/ml.

Alveolar ridge augmentation using implants coated with recombinant human bone morphogenetic protein‐7 (rhBMP‐7/rhOP‐1): histological observations

BACKGROUND Pre-clinical studies have shown that recombinant human bone morphogenetic protein-2 (rhBMP-2) coated onto purpose-designed titanium porous-oxide surface implants induces clinically relevant bone formation and osseointegration. The objective of this study was to examine the potential of rhBMP-7, also known as recombinant human osteogenic protein-1 (rhOP-1), coated onto titanium porous-oxide surface implants to support vertical alveolar ridge augmentation and implant osseointegration. MATERIALS AND METHODS Bilateral, critical-size, 5 mm, supraalveolar peri-implant defects were created in six young adult Hound Labrador mongrel dogs. The animals received implants coated with rhBMP-7 at 1.5 or 3.0 mg/ml randomized to contra-lateral jaw quadrants. The mucoperiosteal flaps were advanced, adapted, and sutured to submerge the implants for primary intention healing. The animals received fluorescent bone markers at 3, 4, 7, and 8 weeks post-surgery when they were euthanized for histological evaluation. RESULTS Without striking differences between treatments, the implant sites exhibited a swelling that gradually regressed to become hard to palpation disguising the implant contours. The histological evaluation showed robust bone formation; the newly formed bone assuming characteristics of the contiguous resident bone, bone formation (height and area) averaging 4.1+/-1.0 versus 3.6+/-1.7 mm and 3.6+/-1.9 versus 3.1+/-1.8 mm(2); and bone density 56%versus 50% for implants coated with rhBMP-7 at 1.5 and 3.0 mg/ml, respectively. Both treatments exhibited clinically relevant osseointegration, the corresponding bone-implant contact values averaging 51% and 47%. Notable peri-implant resident bone remodelling was observed for implants coated with rhBMP-7 at 3.0 mg/ml. CONCLUSIONS rhBMP-7 coated onto titanium porous-oxide surface implants induces clinically relevant local bone formation including osseointegration and vertical augmentation of the alveolar ridge, the higher concentration/dose associated with some local side effects.

Endothelial microvascular networks affect gene-expression profiles and osteogenic potential of tissue-engineered constructs.

IntroductionA major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene-expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation.MethodsMSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining.ResultsThe results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks.ConclusionsThis study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs.

Er:YAG laser in the treatment of periodontal sites with recurring chronic inflammation: a 12‐month randomized, controlled clinical trial

AIM The objective of this randomized, controlled clinical trial was to compare the clinical and microbiological effects of pocket debridement using erbium-doped: yttrium, aluminium and garnet (Er:YAG) laser with conventional debridement in maintenance patients. MATERIAL & METHODS Fifteen patients, all smokers, having at least four teeth with residual probing depth (PD) ≥ 5 mm were recruited. Two pockets in two jaw quadrants were randomly assigned to subgingival debridement using an Er:YAG laser (test) or ultrasonic scaler/curette (control) at 3-month intervals. Relative attachment level (RAL), PD, bleeding on probing and dental plaque were recorded at baseline and at 6 and 12 months. Microbiological subgingival samples were taken at the same time points and analysed using a checkerboard DNA-DNA hybridization technique. RESULTS A significant decrease in PD took place in both treatments from baseline to 12 months (p < 0.01). In the control, the mean initial PD decreased from 5.4 to 4.0 mm at 12 months. For the test, a similar decrease occurred. No significant between-treatment differences were shown at any time point. The mean RAL showed no overall significant inter- or intra-treatment differences (p > 0.05). No significant between-treatment differences were observed in subgingival microbiological composition or total pathogens. CONCLUSION The results failed to support that an Er:YAG laser may be superior to conventional debridement in the treatment of smokers with recurring chronic inflammation. This appears to be the first time that repeated Er-YAG laser instrumentation has been compared with mechanical instrumentation of periodontal sites with recurring chronic inflammation over a clinically relevant time period.

Mapping of titanium particles in peri-implant oral mucosa by laser ablation inductively coupled plasma mass spectrometry and high-resolution optical darkfield microscopy.

The present study examines the quantity, size, element signatures and distribution of titanium particles in normal oral mucosal tissue and in oral mucosa exposed to a titanium implant. Tissue samples from six healthy patients were obtained by a full thickness biopsy taken from the edge of the oral mucosa when inserting a titanium dental implant. At the abutment insertion 6 months later, a punch test biopsy of oral mucosa was taken over the implant site. Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) is a sensitive and specific multi-element microanalytical technique that demonstrated the presence of Ti particles in the tissue adjacent to implant cover-screws. The epithelial part of the control samples revealed more particles than the corresponding area of the test samples, consisting partly of newly formed scar tissue. High-Resolution Optical Darkfield Microscope (HR-ODM) confirmed the presence of particles in both the control and the test samples. The combination of LA-ICP-MS and HR-ODM appears to be a powerful combination for detection of particles in oral tissues; optical microscopy provides an overview with histological references, whereas LA-ICP-MS identifies the chemical composition.

Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells

To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM) was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering.

Site-specific treatment outcome in smokers following non-surgical and surgical periodontal therapy.

Abstract Aim To evaluate the effect of smoking at patient, tooth, and site level following non‐surgical and surgical periodontal therapy. Material and Methods Eighty chronic periodontitis patients, 40 smokers and 40 non‐smokers, were recruited to this single‐arm clinical trial. Smoking status was validated by measuring serum cotinine levels. Periodontal examinations were performed at baseline (T0) and 3 months following non‐surgical and surgical periodontal therapy (T1). At T0 and T1, subgingival plaque samples were collected from the deepest periodontal pocket in each patient and analysed using checkerboard DNA–DNA hybridization. Probing depth (PD) ≥ 5 mm with bleeding on probing (BoP) was defined as the primary outcome. Unadjusted and adjusted logistic regression analyses, corrected for clustered observations within patients and teeth, were conducted comparing smokers with non‐smokers. Results Clinical parameters significantly improved in both groups (p < 0.001). An association was revealed between smoking and PD ≥ 5 mm with BoP (OR= 1.90, CI: 1.14, 3.15, p = 0.013), especially for plaque‐positive sites (OR= 4.14, CI: 2.16, 7.96, p < 0.001). A significant reduction of red complex microbiota was observed for non‐smokers only (p = 0.010). Conclusion Smokers respond less favourably to non‐surgical and surgical periodontal therapy compared with non‐smokers, in particular at plaque‐positive sites.

Alveolar ridge augmentation using implants coated with recombinant human growth/differentiation factor -5 (rhGDF-5). Radiographic observations

OBJECTIVES Application of growth factors onto dental implant surfaces is being considered to support local bone formation. Bone morphogenetic protein-2 (BMP-2) and BMP-7 have been shown to support local bone formation, but are also associated with adverse events including seroma formation, extensive bone remodeling, and implant displacement captured in the radiographic evaluation. This report presents mineralized tissue formation and associated adverse events following implantation of recombinant human growth/differentiation factor-5 (rhGDF-5) coated onto a purpose-designed titanium porous-oxide implant surface. MATERIAL AND METHODS Twelve young adult Labrador dogs were used. Three 10-mm titanium implants/jaw quadrant were placed 5 mm into the alveolar ridge in the posterior mandible following surgical extraction of the premolar teeth and reduction of the alveolar ridge. Six animals received implants coated with rhGDF-5 at 30 or 60 μg/implant in contralateral jaw quadrants. Six animals received implants coated with rhGDF-5 at 120 μg/implant or uncoated implants (sham-surgery control) using the same split-mouth design. The mucoperiosteal flaps were advanced, adapted, and sutured to submerge the implants. Radiographic recordings were made immediately postsurgery (baseline), and at week 4 and 8 (end of study). Two masked examiners performed the analysis using computer enhanced radiographic images. RESULTS rhGDF-5 coated implants displayed mineralized tissue formation significantly exceeding that of the sham-surgery control in a dose-dependent order. The greatest increase was observed for implants coated with rhGDF-5 at 60 μg and 120 μg amounting to approximately 2.2 mm for both groups at 8 weeks. Importantly, none of the implants showed evidence of peri-implant bone remodeling, implant displacement, or seroma formation. The newly formed mineralized tissues assumed characteristics of the resident bone. CONCLUSIONS rhGDF-5 coated onto a titanium porous-oxide implant surface exhibits a dose-dependent potential to stimulate local mineralized tissue formation. Application of rhGDF-5 appears safe as it is associated with limited, if any, adverse events.

Cell seeding density is a critical determinant for copolymer scaffolds-induced bone regeneration

Abstract Constructs intended for bone tissue engineering (TE) are influenced by the initial cell seeding density. Therefore, the objective of this study was to determine the effect of bone marrow stromal stem cells (BMSCs) density loaded onto copolymer scaffolds on bone regeneration. BMSCs were harvested from rats bone marrow and cultured in media with or without osteogenic supplements. Cells were seeded onto poly(l‐lactide‐co‐ε‐caprolactone) [poly(LLA‐co‐CL)] scaffolds at two different densities: low density (1 × 106 cells/scaffold) or high density (2 × 106 cells/scaffold) using spinner modified flasks and examined after 1 and 3 weeks. Initial attachment and spread of BMSC onto the scaffolds was recorded by scanning electron microscopy. Cell proliferation was assessed by DNA quantification and cell differentiation by quantitative real‐time reverse transcriptase‐polymerized chain reaction analysis (qRT‐PCR). Five‐millimeter rat calvarial defects (24 defects in 12 rats) were implanted with scaffolds seeded with either low or high density expanded with or without osteogenic supplements. Osteogenic supplements significantly increased cell proliferation (p < 0.001). Scaffolds seeded at high cell density exhibited higher mRNA expressions of Runx2 p = 0.001, Col1 p = 0.001, BMP2 p < 0.001, BSP p < 0.001, and OC p = 0.013. More bone was formed in response to high cell seeding density (p = 0.023) and high seeding density with osteogenic medium (p = 0.038). Poly (LLA‐co‐CL) scaffolds could be appropriate candidates for bone TE. The optimal number of cells to be loaded onto scaffolds is critical for promoting Extracellular matrix synthesis and bone formation. Cell seeding density and osteogenic supplements may have a synergistic effect on the induction of new bone.

A Copolymer Scaffold Functionalized with Nanodiamond Particles Enhances Osteogenic Metabolic Activity and Bone Regeneration.

Functionalizing polymer scaffolds with nanodiamond particles (nDPs) has pronounced effect on the surface properties, such as improved wettability, an increased active area and binding sites for cellular attachment and adhesion, and increased ability to immobilize biomolecules by physical adsorption. This study aims to evaluate the effect of poly(l-lactide-co-ε-caprolactone) (poly(LLA-co-CL)) scaffolds, functionalized with nDPs, on bone regeneration in a rat calvarial critical size defect. Poly(LLA-co-CL) scaffolds functionalized with nDPs are also compared with pristine scaffolds with reference to albumin adsorption and seeding efficiency of bone marrow stromal cells (BMSCs). Compared with pristine scaffolds, the experimental scaffolds exhibit a reduction in albumin adsorption and a significant increase in the seeding efficiency of BMSCs (p = 0.027). In the calvarial defects implanted with BMSC-seeded poly(LLA-co-CL)/nDPs scaffolds, live imaging at 12 weeks discloses a significant increase in osteogenic metabolic activity (p = 0.016). Microcomputed tomography, confirmed by histological data, reveals a substantial increase in bone volume (p = 0.021). The results show that compared with conventional poly(LLA-co-CL) scaffolds those functionalized with nDPs promote osteogenic metabolic activity and mineralization capacity. It is concluded that poly(LLA-co-CL) composite matrices functionalized with nDPs enhance osteoconductivity and therefore warrant further study as potential scaffolding material for bone tissue engineering.