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Tissue engineering generally requires three basic elements; stem/progenitor cells, inductive agents and a biomaterial scaffold; the latter is one of the key components which directly influences cellular activity and matrix formation. Commonly used scaffolds to repair defects in general do not induce stem cell recruitment, which is an essential element to tissue regeneration. In this study, fabrication of a scaffold which is capable of restoring damaged tissue through the recruitment of mesenchymal stem cells (MSCs) by gene therapy of the gene encoding platelet-derived growth factor-B (PDGF-B) was investigated. PDGF-B adenovirus (AdPDGF) was combined into novel mesoporous bioglass-silk fibrin scaffolds, which were characterized for their controlled release and sustained bioactivity. Our results demonstrate that these scaffolds can release PDGF-B adenovirus for up to 3 weeks and increase MSC recruitment, both in vitro and following subcutaneous implantation in mice. Osseous calvarial defects in mice further demonstrate the ability of these scaffolds to enhance tissue regeneration through stem cell homing. This study demonstrates the potent ability of host stem cells to regenerate tissue defects through recruitment of MSCs via gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.

Objective The purpose of this investigation was to compare the osteoinductive and osteopromotive potential of two widely used demineralized freeze-dried bone allografts (DFDBA) (Osteotech® DFDBA and LifeNet® DFDBA). Material and Methods Twenty-seven male Wistar rats (mean body weight 200 g) were treated with either DFDBA from Osteotech and LifeNet or control for femoral and intramuscular defects and assigned to histological analysis at 2, 4, and 8 weeks postimplantation. Hematoxylin and eosin (H&E), Safranin-O, tartrate-resistant acid phosphatase (TRAP), and osteopontin (OPN) staining were performed. Quantitative analysis of mineralized new bone to total volume (BV/TV) was assessed by micro-computed tomography. Results Both allografts demonstrated osteoinductive potential at 2 weeks as assessed by intramuscular bone formation. LifeNet DFDBA displayed continual new bone formation at 4 and 8 weeks, whereas Osteotech particles were fully resorbed by 4 weeks postimplantation. Femur defects demonstrated significantly greater BV/TV at 4 and 8 weeks with higher expression of OPN staining around LifeNet DFDBA particles. TRAP-positive cells were visible in and around both allograft materials. Conclusion The results from the present study indicate that variability among allografts exists. In the present, LifeNet DFDBA supported more new bone formation. Further larger animal models or clinical trials are required to validate these findings.

The most common complication for patients with postmenopausal osteoporosis is bone-related defects and fractures. While routine medication has a high probability of undesirable side effects, new approaches have aimed to develop regeneration procedures that stimulate new bone formation while reversing bone loss. Recently, we have synthesized a new hybrid CaP/silk scaffold with a CaP-phase distribution and pore architecture better suited to facilitate cell differentiation and bone formation. The aim of the present study was to compare the involved remodeling process and therapeutic effect of porous CaP/silk composite scaffolds upon local implantation into osteoporotic defects. Wistar rats were used to induce postmenopausal osteoporotic model by bilateral ovariectomy. The pure silk and hybrid CaP/silk scaffolds were implanted into critical sized defects created in distal femoral epiphysis. After 14 and 28 days, the in vivo osteogenetic efficiency was evaluated by μCT analysis, hematoxylin and eosin staining, Safranin O staining, tartrate-resistant acid phosphatase staining, and immunohistochemical assessment. Animals with or without critical-sized defects were used as drill or blank controls, respectively. The osteoporotic defect model was well established with significantly decreased μCT parameters of BV/TV, Tb.N and increased Tb.Sp, porosity, combined with changes in histological observations. During the healing process, the critical-sized drill control defects failed to regenerate appreciable bone tissue, while more significantly increased bone formation and mineralization with dynamic scaffold degradation and decreased osteoclastic bone resorption could be detected within defects with hybrid CaP/silk scaffolds compared to pure silk scaffolds.

The purpose of this study was to evaluate the effect of early functional loading to plateaued implants on bone formation and implant stability in a dog model.Early loading (EL), nonloading control, and delayed loading (DL) groups were compared using six beagle dogs under functional loading. The Periotest® values were measured dynamically for 6 weeks. Peri-implant bone architecture was evaluated qualitatively by microcomputed tomography (μCT) and analyzed quantitatively by mineral apposition rates (MAR), bone-to-implant contact (BIC), and bone volumes (BV/TV) after the euthanasia at 3 and 6 weeks after loading.The EL implants showed poor stability at 1 week, but greater stability at 2 and 4 weeks after loading compared to DL implants. There was no significant difference between MAR of EL and unloaded implants at both time intervals. The EL implants displayed a significantly higher MAR when compared to DL implants at 3-5 weeks. A significantly higher BIC for the DL group was observed when compared to the EL group at 3 weeks following loading, however at 6 weeks; no significant difference between these groups was observed. The EL group gained a higher BIC than the no-treatment control group at 6 weeks.For plateaued implant, the decreased healing time (1 week) displays a positive effect on peri-implant bone (re)modeling under functional loading during the early phase.The early application of functional loading on plateaued implants can be used clinically to shorten the course of treatment and improve esthetics.

Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze-dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells.DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining.Analysis of cell attachment revealed no significant differences among control, blood-coated, and EMD-coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood-coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD-coated DFDBA particles at 3, 7, and 14 days.The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.

Osteoporosis is one of the most widely occurring bone disorders characterized by low bone mineral density and poor bone strength. Strontium ranelate, as a treatment option, has received significant attention in recent years due to its ability to halt the progress of osteoporosis by simultaneously improving bone formation and reducing bone resorption. Although much emphasis has been given to the treatment of osteoporosis and fracture prevention using pharmacological agents, much less attention has been placed on the repair of critical-sized bone fractures caused by osteoporosis. The aim of the present study was to prepare strontium-incorporated mesoporous bioactive glass (Sr-MBG) scaffolds in order to combine the therapeutic effects of Sr2+ ions on osteoporosis with the bioactivity of MBG to regenerate osteoporotic-related fractures. Prior to animal implantation, the effects of Sr-containing ionic products from Sr-MBG scaffolds on the proliferation and differentiation of bone marrow stromal cells (BMSCs) from osteoporotic bone were investigated in an in vitro culture system. The results showed that Sr-MBG scaffolds significantly increased the proliferation of BMSCs in a concentration dependent manner and were able to stimulate the expression of osteoblast differentiation markers including Alpl, Col1a1, Runx2 and Bglap as assessed by real-time PCR. Critical sized femur defects in ovariectomised rats were created to simulate an osteoporotic phenotype. At time points 2, 4 and 8 weeks post-implantation, the in vivo osteogenetic efficiency was systematically evaluated by μCT analysis, hematoxylin and eosin staining, and immunohistochemistry (type I collagen). The results showed that the incorporation of Sr into MBG scaffolds significantly stimulated new bone formation in osteoporotic bone defects when compared to MBG scaffolds alone. Furthermore, it was generally found that Sr release in blood was maintained at a very low level and the Sr, Si, Ca and P excretion by urine operated in an a similar manner to blank control animals. Our results suggested that Sr-MBG scaffolds could be a promising biomaterial for regenerating osteoporosis-related fractures by the release of Sr-containing ionic products.

OBJECTIVES The aim of the present study was to investigate the effects of Osteogain, a new formulation of enamel matrix derivative (EMD) in combination with a grafting material on a wide variety of genes for cytokines, transcription factors and extracellular matrix proteins involved in osteoblast differentiation. MATERIALS AND METHODS Primary human periodontal ligament (PDL) cells were seeded on natural bone mineral (NBM) particles coated with Osteogain for 24 h and analyzed for regulated gene expression using a human osteogenesis gene super-array kit. Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation as well as gene products representing extracellular matrix molecules, transcription factors and cell adhesion molecules. RESULTS Osteogain significantly upregulated the expression of over 20 of the 100 genes examined including bone morphogenetic protein 2 (BMP2), TGFβ1, fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) as well as some of their associated receptors. Osteogain also promoted gene expression of a number of osteoblast differentiation markers including collagen1α2 and alkaline phosphatase as well as cell adhesion molecules including fibronectin and a variety of integrin binding proteins. Interestingly, Osteogain promoted calcitonin receptor 55-fold and also promoted annexin A5 gene expression over 12-fold. CONCLUSION The present study demonstrates that Osteogain is capable of either upregulating or downregulating the expression of a wide variety of genes including those for growth factors and cytokines when combined with a bone grafting material. CLINICAL RELEVANCE The results from the present study demonstrate the large and potent effect of addition of Osteogain in combination to a bone grafting material over a wide variety of genes supporting osteogenesis.

OBJECTIVES Among bone morphogenetic protein (BMP) family members, BMP-2 and BMP-9 have demonstrated potent osteoinductive potential. However, in vivo differences in their potential for bone regeneration remain unclear. The present study aimed to compare the effects of recombinant human (rh) BMP-2 and rhBMP-9 on bone formation in rat calvarial critical-size defects (CSD). MATERIALS AND METHODS Twenty-eight Wistar rats surgically received two calvarial defects bilaterally in each parietal bone. Defects (n = 56) were allocated into four groups: absorbable collagen sponge (ACS) alone, rhBMP-2 with ACS (rhBMP-2/ACS), rhBMP-9/ACS, or sham surgery (control), on the condition that the treatments of rhBMP-2/ACS and rhBMP-9/ACS, or the same treatments were not included in the same animal. Animals were sacrificed at 2 and 8 weeks post-surgery. The calvarial defects were analyzed for bone volume (BV) by micro-computed tomography and for percentages of defect closure (DC/DL), newly formed bone area (NBA/TA), bone marrow area (BMA/NBA), adipose tissue area (ATA/NBA), central bone height (CBH), and marginal bone height (MBH) by histomorphometric analysis. RESULTS The BV in the rhBMP-2/ACS group (5.44 ± 3.65 mm3, n = 7) was greater than the other groups at 2 weeks post-surgery, and the rhBMP-2/ACS and rhBMP-9/ACS groups (18.17 ± 2.51 and 16.30 ± 2.46 mm3, n = 7, respectively) demonstrated significantly greater amounts of BV compared with the control and ACS groups (6.02 ± 2.90 and 9.30 ± 2.75 mm3, n = 7, respectively) at 8 weeks post-surgery. The rhBMP-2/ACS and rhBMP-9/ACS groups significantly induced new bone formation compared to the control and ACS groups at 8 weeks post-surgery. However, there were no statistically significant differences found between the rhBMP-2/ACS and rhBMP-9/ACS groups in any of the histomorphometric parameters. The ATA/NBA in the rhBMP-2/ACS group (9.24 ± 3.72%, n = 7) was the highest among the treatment groups at 8 weeks post-surgery. CONCLUSIONS Within the limits of this study, it can be concluded that rhBMP-2/ACS induced a slight early increase in new bone formation at 2 weeks and that rhBMP-9/ACS provided comparable new bone formation to rhBMP-2/ACS with less adipose tissues after a healing period of 8 weeks in rat CSD. CLINICAL RELEVANCE RhBMP-9/ACS treatment provided new bone formation with less adipose tissues compared with rhBMP-2/ACS.

The delivery of novel bioactive scaffolds for the repair of bone defects remains a prominent challenge worldwide. Currently osteoporosis, a disease caused by low bone mineral density affects over 200 million people worldwide with up to half of this population experiencing at least one fracture within their lifetime. Recently temperature-sensitive p(N-isopropylacrylamide-co-butyl methylacrylate) nanogel (PIB nanogel) scaffolds have emerged as biomaterial candidate for regenerative therapies. It has the advantage of being injected from syringes as a soluble gel form (capable of delivering growth and/or living progenitor cells) yet hardens once it reaches body temperatures. Although this material demonstrates optimal clinical delivery of scaffolds, its main drawback is its low osteoconductivity and bioactivity. Recently we have demonstrated that mesoporous bioactive glass (MBG) loaded with strontium was able to regenerate osteoporotic defects in vivo and enhance osteoblast differentiation in vitro. The aim of this study was to combine the advantages of these two therapies and prepare PIB-nanogel scaffolds containing Sr-MBG and investigate their ability to regenerate femur defects created in ovarectamized rats. The results demonstrate that groups containing Sr-MBG within the nanogel formulation had significantly higher new bone formation when compared with other modalities. We further demonstrate that although nanogel demonstrated poor osteogenic ability, the addition of osteoblasts worked synergistically with Sr-MBG particles to enhance the regeneration of the created femur defects in osteoporotic animals. In conclusion, PIB nanogel scaffolds are a viable treatment modality for bone tissue engineering and may serve as a carrier-scaffold for osteogenic cells and/or bioactive scaffolds such as Sr-MBG. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1175-1183, 2017.

OBJECTIVES: Dimensional changes of the alveolar bone following tooth extraction are a major challenge in daily dental practice. To limit bone loss, a variety of biomaterials including bone grafts, barrier membranes, and growth factors have been utilized either alone or in combination therapies to increase the speed and quality of new bone formation. The aim of the present in vitro study was to investigate the regenerative potential of Osteogain®, a new liquid carrier system of enamel matrix derivative (EMD) in combination with an absorbable collagen sponge (ACS) specifically designed for extraction socket healing. MATERIALS AND METHODS: The potential of ACS was first investigated using ELISA to quantify total amelogenin adsorption and release from 0 to 10 days. Thereafter, the cellular effects of ST2 pre-osteoblasts were investigated for cellular attachment at 8 h and cell proliferation at 1, 3, and 5 days as well as osteoblast differentiation by real-time PCR and alizarin red staining for cells seeded on (1) tissue culture plastic, (2) ACS alone, and (3) ACS + Osteogain®. RESULTS: ACS efficiently loaded nearly 100% of the amelogenin proteins found in Osteogain® which were gradually released up to a 10-day period. Osteogain® also significantly induced a 1.5-fold increase in cell attachment and resulted in a 2-6-fold increase in mRNA levels of osteoblast differentiation markers including runt-related transcription factor 2 (Runx2), collagen1a2, alkaline phosphatase, and bone sialoprotein as well as induced alizarin red staining when combined with ACS. CONCLUSIONS: In summary, these findings suggest that Osteogain® is capable of inducing osteoblast attachment and differentiation when combined with ACS. Future animal studies and randomized human clinical trials are necessary to further support these findings. CLINICAL RELEVANCE: The use of Osteogain® in combination with ACS may provide a valuable means to limit dimensional changes following tooth extraction. KEYWORDS: Absorbable collagen sponge; Bone regeneration; Enamel matrix derivative; Enamel matrix proteins; Growth factor; Regenerative therapy; Tooth extraction; Tooth loss

The growing multidisciplinary field of tissue engineering aims at predictably regenerating, enhancing, or replacing damaged or missing tissues for a variety of conditions caused by trauma, disease, and old age. One area of research that has gained tremendous awareness in recent years is that of platelet-rich fibrin (PRF), which has been utilized across a wide variety of medical fields for the regeneration of soft tissues. This systematic review gathered all the currently available in vitro, in vivo, and clinical literature utilizing PRF for soft tissue regeneration, augmentation, and/or wound healing. In total, 164 publications met the original search criteria, with a total of 48 publications meeting inclusion criteria (kappa score = 94%). These studies were divided into 7 in vitro, 11 in vivo, and 31 clinical studies. In summary, 6 out of 7 (85.7%) and 11 out of 11 (100%) of the in vitro and in vivo studies, respectively, demonstrated a statistically significant advantage for combining PRF to their regenerative therapies. Out of the remaining 31 clinical studies, a total of 8 reported the effects of PRF in a randomized clinical trial, with 5 additional studies (13 total) reporting appropriate controls. In those clinical studies, 9 out of the 13 studies (69.2%) demonstrated a statistically relevant positive outcome for the primary endpoints measured. In total, 18 studies (58% of clinical studies) reported positive wound-healing events associated with the use of PRF, despite using controls. Furthermore, 27 of the 31 clinical studies (87%) supported the use of PRF for soft tissue regeneration and wound healing for a variety of procedures in medicine and dentistry. In conclusion, the results from the present systematic review highlight the positive effects of PRF on wound healing after regenerative therapy for the management of various soft tissue defects found in medicine and dentistry.

Extracorporeal irradiation sterilizes resected tumor bone used as autografts in reconstruction surgery. Therapeutic irradiation is a standard technique in head and neck cancer therapy that aims to preserve organ function. Bone irradiation has a complex, mostly inhibitory, effect on remodeling and regeneration, although the underlying mechanisms are still not fully understood. It remains unclear if extracorporeal irradiation affects the paracrine-like activity of the corresponding autografts. We recently reported that bone-conditioned medium from autogenous bone chips contains a number of factors that might affect cell activity. In the present study, we investigated the effects of extracorporeal irradiation of porcine cortical bone chips on the activity of the corresponding bone-conditioned medium. The effects of bone-conditioned medium on the expressions of transforming growth factor-beta (TGF-β) target genes in oral fibroblasts were assessed. Bone-conditioned medium from bone chips exposed to a total radiation dose up to 120 Gy did not affect expressions of TGF-β target genes, including adrenomedullin, BTB/POZ domain-containing protein 11, proteoglycan 4, NADPH oxidase 4, and interleukin 11, in oral fibroblasts. In conclusion, bone irradiation does not alter the capability of the corresponding bone-conditioned medium to provoke a robust fibroblastic cell response in vitro. (J Oral Sci 58, 325-331, 2016).

Within the past years, BMP9 has been characterized as one of the most osteogenic bone-inducers among the BMP family, however up until recently, BMP9 has only been available through adenovirus transfection experiments (gene therapy) not approved for clinical use. The aim of this study was to investigate recombinant rhBMP9 versus rhBMP2 at 2 concentrations (10 and 100 ng/mL) in combination with 2 bone grafts: (1) a natural bone mineral (NBM) without collagen versus (2) a novel NBM integrated with atelo-collagen type I (NBM-Col). Scanning electron microscopy revealed that while NBM demonstrated a mineralized roughened surface morphology, NBM-Col particles contained many more visible collagen fibrils throughout the scaffold surface significantly increasing rhBMP adsorption from 8 h to 10 days (as quantified by ELISA). Thereafter, ST2 preosteoblasts were used to investigate cell attachment, proliferation, and differentiation. While little change was observed for cell attachment/proliferation, osteoblast differentiation demonstrated a significant increase in alkaline phosphatase (ALP) activity when scaffolds were loaded with rhBMP9 when compared to rhBMP2. Furthermore, a 2-3 fold increase in alizarin red staining, and in mRNA levels of osteoblast differentiation markers Runx2, Collagen1α2, ALP, and osteocalcin was observed when rhBMP9 was combined with NBM-Col when compared to NBM without collagen at equivalent doses and when compared to rhBMP2. The results from this study demonstrate that (1) the use of rhBMP9 significantly and markedly induced osteoblast differentiation when compared to rhBMP2 and (2) the incorporation of atelo-collagen type I into NBM bone grafts markedly improved these findings by serving as a scaffold capable of improving growth factor adsorption and osteoblast behavior. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 408-418, 2017.

OBJECTIVES This study investigated the effect of smooth and rough titanium surface topographies on macrophage polarization and their influence on gingival fibroblast behavior cultured on titanium surfaces. MATERIALS AND METHODS RAW 264.7 macrophages were seeded on smooth (pickled titanium (PT)) and rough Sand-blasted with Large grit particles followed by Acid-etching (SLA) titanium surfaces and first investigated for macrophage polarization towards tissue-inflammatory M1 macrophages or wound-healing M2 macrophages. Thereafter, culture media collected from macrophages on both surfaces were cultured with gingival fibroblasts seeded on their respective topographies. All experiments were performed in triplicate with three independent experiments. RESULTS Macrophages seeded on SLA surfaces polarized towards tissue-inflammatory M1 macrophages at early time points. Immunofluorescent staining and RT-PCR analysis demonstrated higher levels of iNOS and gene expression of IL-1β, IL-6, and TNF-alpha on SLA surfaces at 3 days when compared to both tissue culture plastic (TCP) and PT surfaces (p

In the craniofacial bone field, fibrin sealants are used as coagulant and adhesive tools to stabilize grafts during surgery. Despite this, their exact role in osteogenesis is poorly characterized. In the present study, we aimed to characterize the osteogenic potential of TISSEEL fibrin sealant and used its technology to incorporate growth factors within its matrix. We focused on recombinant human bone morphogenetic protein (rhBMP)–9, which has previously been characterized as one of the strongest osteogenetic inducers in the BMP family. TISSEEL displayed an excellent ability to retain rhBMP9, which was gradually released over a 10-day period. Although TISSEEL decreased bone stromal ST2 cell attachment at 8 h, it displayed normal cell proliferation at 1, 3, and 5 days when compared to tissue culture plastic. Interestingly, TISSEEL had little influence on osteoblast differentiation; however its combination with rhBMP9 significantly increased ALP activity at 7 days, Alizarin Red staining at 14 days, and mRNA levels of osteoblast differentiation markers ALP, bone sialoprotein, and osteocalcin. In summary, although fibrin sealants were shown to have little influence on osteogenesis, their combination with bone-inducing growth factors such as rhBMP9 may serve as an attractive carrier/scaffold for future bone regenerative strategies. Future animal studies are now necessary.

BACKGROUND High dose radiation therapy is commonly used in maxillofacial surgeries to treat a number of head and neck tumors. Despite its widespread use, little information is available regarding the effects of irradiation on bone cell viability and release of growth factors following dose-dependent irradiation. METHODS Bone samples were collected from porcine mandibular cortical bone and irradiated at doses of 0, 7.5, 15, 30, 60 and 120 Grays. Thereafter, cell viability was quantified, and the release of growth factors including TGFβ1, BMP2, VEGF, IL1β and RANKL were investigated over time. RESULTS It was observed that at only 7.5Gy of irradiation, over 85 % of cells were non-vital and by 60 Gy, all cells underwent apoptosis. Furthermore, over a 7-fold decrease in VEGF and a 2-fold decrease in TGFβ1 were observed following irradiation at all tested doses. Little change was observed for BMP2 and IL1β whereas RANKL was significantly increased for all irradiated samples. CONCLUSIONS These results demonstrate the pronounced effects of irradiation on bone-cell vitality and subsequent release of growth factors. Interestingly, the largest observed change in gene expression was the 7-fold decrease in VEGF protein following irradiation. Future research aimed at improving our understanding of bone following irradiation is necessary to further improve future clinical treatments.

BACKGROUND The use of autogenous bone chips during guided bone regeneration procedures has remained the gold standard for bone grafting due to its excellent combination of osteoconduction, osteoinduction and osteogenesis. Recent protocols established by our group have characterized specific growth factors and cytokines released from autogenous bone that have the potential to be harvested and isolated into bone conditioned media (BCM). Due to the advantageous osteo-promotive properties of BCM, the aims of the present study was to pre-coat collagen barrier membranes with BCM and investigate its effect on osteoblast adhesion, proliferation and differentiation for possible future clinical use. METHODS Scanning electron microscopy (SEM) was first used to qualitative assess BCM protein accumulation on the surface of collagen membranes. Thereafter, undifferentiated mouse ST2 stromal bone marrow cells were seeded onto BioGide porcine derived collagen barrier membranes (control) or barrier membranes pre-coated with BCM (test group). Control and BCM samples were compared for cell adhesion at 8 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 5 days for osteoblast differentiation markers including Runx2, alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP). Mineralization was further assessed with alizarin red staining at 14 days post seeding. RESULTS SEM images demonstrated evidence of accumulated proteins found on the surface of collagen membranes following coating with BCM. Analysis of total cell numbers revealed that the additional pre-coating with BCM markedly increased cell attachment over 4 fold when compared to cells seeded on barrier membranes alone. No significant difference could be observed for cell proliferation at all time points. BCM significantly increased mRNA levels of osteoblast differentiation markers including ALP, OCN and BSP at 5 days post seeding. Furthermore, barrier membranes pre-coated with BCM demonstrated a 5-fold increase in alizarin red staining at 14 days. CONCLUSION The results from the present study suggest that the osteoconductive properties of porcine-derived barrier membranes could be further improved by BCM by significantly increasing cell attachment, differentiation and mineralization of osteoblasts in vitro. Future animal testing is required to fully characterize the additional benefits of BCM for guided bone regeneration.

Osteoporosis is a prominent disorder affecting over 200 million people worldwide. Recently, semaphorins have been implicated in the cell-cell communication between osteoclasts and osteoblasts and have been associated with the progression of osteoporosis. Previously, we demonstrated that knockdown of semaphorin4d (Sema4d) using siRNA delivered with a bone-targeting system prevented bone loss in an osteoporotic animal model. Here, we used this bone-specific technology containing siRNA-Sema4d and fabricated a PLLA scaffold capable of enhancing bone repair following fracture. We investigated the ability of the implant to release siRNA-Sema4d into the surrounding tissues over time and to influence new bone formation in a 3 mm femur osteoporotic defect model in ovariectomized rats. Delivery of the bone-targeting system released from PLLA scaffolds began 2 hours post-implantation, peaked at 1 day and was sustained over a 21 day period. μCT analysis demonstrated a significantly higher bone volume/total volume bone mineral density and number of osteoblasts in the rats that were transplanted with scaffolds loaded with siRNA-Sema4d. These results confirm the specific role of Sema4d in bone remodeling and demonstrate that significant increases in the speed and quality of new bone formation occur when siRNA-Sema4d is delivered via a PLLA scaffold.

OBJECTIVE Although an extensive amount of research has demonstrated the positive effects of an enamel matrix derivative (EMD) on soft tissue wound healing around intrabony defects, little information is available describing its effect on peri-implant soft tissues, an area that has recently gained tremendous awareness due to the increasing prevalence of peri-implantitis. The aim of the present study was to assess the role of EMD when gingival fibroblasts were cultured on titanium surface with different surface topographies. METHODS Human primary gingival fibroblasts were cultured on pickled (PT) and sand-blasted with large grit followed by acid etching (SLA) surfaces and assessed for cell adhesion at 2, 4, and 8 h, cell morphology at 2, 4, 8, and 24 h as well as cell proliferation at 1, 3, and 5 days post-seeding. Furthermore, genes encoding collagen 1a1, vascular endothelial growth factor-A (VEGF-A), and fibronectin were assessed by real-time PCR. Human gingival fibroblasts were also quantified for their ability to synthesize a collagen matrix on the various titanium surfaces with and without EMD by immunofluorescence staining. RESULTS The results from the present study demonstrate that EMD significantly increased cell spreading at 2, 4, 8, and 24 h on PT surfaces and 4, 8, and 24 h on SLA surfaces. Furthermore, proliferation at 5 days on PT surfaces and 3 and 5 days on SLA surfaces was also increased for groups containing EMD. Real-time PCR results demonstrated that the culture of gingival fibroblasts with EMD significantly increased extracellular matrix synthesis of collagen 1 as well as improved mRNA levels of VEGF-A and fibronectin. Collagen1 immuno-fluorescent staining revealed a significantly higher area of staining for cells seeded on PT + EMD at 7 and 14 days and 14 days for SLA + EMD when compared to control samples. CONCLUSION The results from the present study favor the use of EMD for colonization of gingival fibroblasts on titanium surfaces by increasing cell growth, spreading, and synthesis of an extracellular matrix. The improvements were primarily irrespective of surface topography. Future animal and human studies are necessary to fully characterize the beneficial effects of incorporating EMD during soft tissue regeneration of implant protocols. CLINICAL RELEVANCE The use of EMD may speed up the quality of soft tissue integration around dental implants by facilitating gingival cell attachment, proliferation, and matrix synthesis of collagen 1.

OBJECTIVES Recently, novel biphasic calcium phosphate (BCP) scaffolds have emerged as a new class of bone grafts with osteoinductive potential demonstrating the ability to form ectopic bone in extra-skeletal sites. The aim of the present study was to perform an osteogenic gene array to target possible genes responsible for eliciting the changes in cell expression responsible for inducing osteoblast differentiation. MATERIALS AND METHODS Human MG63 osteoblast-like cells were seeded for 24 h on tissue culture plastic or osteoinductive BCP particles and analyzed for upregulated genes using an osteogenesis super-array. Osteoblast-related genes including those transcribed during bone mineralization, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules were investigated. RESULTS An upregulation of genes transcribing biglycan (1.7-fold), bone morphogenetic proteins 1, 2, 4, 6, and 7 (1.5-2.1-fold), various collagen isoforms including 1a1, 1a2, 2a1, and 5a1 (1.73-2.72-fold), colony stimulating factor 2 (2.59-fold), fibroblast growth factor receptor 2 (1.79-fold), fibronectin (2.56-fold), integrin alpha 1, 2, and 3 (1.82-2.24-fold), SOX9 (2.75-fold), transforming growth factor beta receptor 2 (1.72-fold), vitamin D (1.89-fold), and vascular endothelial growth factor A and B (2.00, 1.75-fold) were all significantly (p

Hyaluronic acid (HA) has been utilized for a variety of regenerative medical procedures due to its widespread presence in connective tissue and perceived biocompatibility. The aim of the present study was to investigate HA in combination with recombinant human bone morphogenetic protein 9 (rhBMP9), one of the most osteogenic growth factors of the BMP family. HA was first combined with rhBMP9 and assessed for the adsorption and release of rhBMP9 over 10 days by ELISA. Thereafter, ST2 pre-osteoblasts were investigated by comparing (1) control tissue culture plastic, (2) HA alone, and (3) HA with rhBMP9 (100 ng/mL). Cellular proliferation was investigated by a MTS assay at one, three and five days and osteoblast differentiation was investigated by alkaline phosphatase (ALP) activity at seven days, alizarin red staining at 14 days and real-time PCR for osteoblast differentiation markers. The results demonstrated that rhBMP9 adsorbed within HA scaffolds and was released over a 10-day period in a controlled manner. While HA and rhBMP9 had little effect on cell proliferation, a marked and pronounced effect was observed for cell differentiation. rhBMP9 significantly induced ALP activity, mRNA levels of collagen1α2, and ALP and osteocalcin (OCN) at three or 14 days. HA also demonstrated some ability to induce osteoblast differentiation by increasing mRNA levels of OCN and increasing alizarin red staining at 14 days. In conclusion, the results from the present study demonstrate that (1) HA may serve as a potential carrier for various growth factors, and (2) rhBMP9 is a potent and promising inducer of osteoblast differentiation. Future animal studies are now necessary to investigate this combination approach in vivo. KEYWORDS: BMP; bone formation; bone induction; dimensional changes; growth factor; guided bone regeneration; hard tissue regeneration; osteoinduction; osteoinductive; regenerative therapy

BACKGROUND The repair of alveolar bone defects with growth factors and bone grafting materials has played a pivotal role in modern dentistry. Recombinant human bone morphogenetic protein-2 (rhBMP2), an osteoinductive growth factor capable of cell recruitment and differentiation towards the osteoblast lineage, has been utilized in combination with various biomaterials to further enhance new bone formation. Recently, a group of novel biphasic calcium phosphate (BCP) bone grafting materials have been demonstrated to possess osteoinductive properties by demonstrating signs of ectopic bone formation. The aim of the present study was to study the effects of rhBMP2 in combination with osteoinductive BCP bone grafts on osteoblast cell behaviour. METHODS MC3T3-E1 pre-osteoblasts were seeded on 1) control tissue culture plastic, 2) 10 mg of BCP alone, 3) 100 ng rhBMP2, and 4) 100 ng rhBMP2+ 10 mg of BCP and analyzed for cell recruitment via a Transwell chamber, proliferation via an MTS assay and differentiation as assessed by alkaline phosphatase (ALP) activity, alizarin red staining and real-time PCR for osteoblast differentiation markers including Runx2, collagen1, ALP, and osteocalcin (OCN). RESULTS rhBMP2 was able to significantly upregulate cell recruitment whereas the addition of BCP as well as BCP alone had no additional ability to improve osteoblast recruitment. Both BCP and rhBMP2 were able to significantly increase cell proliferation at 3 and 5 days post seeding and cell number was further enhanced when rhBMP2 was combined with BCP. In addition, the combination of rhBMP2 with BCP significantly improved ALP activity at 7 and 14 days post seeding, alizarin red staining at 14 days, and mRNA levels of Runx2, ALP and osteocalcin when compared to cells seeded with rhBMP2 alone or BCP alone. CONCLUSIONS The results from the present study demonstrate that 1) the osteoinductive potential of BCP bone particles is equally as osteopromotive as rhBMP2 on in vitro osteoblast differentiation and 2) BCP particles in combination with rhBMP2 is able to further increase the osteopromotive differentiation of osteoblasts in vitro when compared to either rhBMP2 alone or BCP alone. Future animal testing is further required to investigate this combination approach on new bone formation.

OBJECTIVES Guided bone regeneration (GBR) aims to predictably restore missing bone that has been lost due to trauma, periodontal disease or a variety of systemic conditions. Critical to this procedure is the ability of a bone grafting material to predictably serve as a 3-dimensional scaffold capable of inducing cell and bone tissue in-growth at the material surface. Although all bone grafts are osteoconductive to bone-forming osteoblasts, only a small number of commercially available bone grafts with FDA approval are osteoinductive including demineralized freeze-dried bone allographs (DFDBA) and scaffolds containing bone morphogenetic proteins (BMPs). Recently, a class of synthetic bone grafts fabricated from biphasic calcium phosphate (BCP) sintered at a low temperature have been shown to form ectopic bone formation in non-skeletal sites without the use of growth factors. Therefore, the present study aimed to compare the osteoinductive potential of this group of synthetic BCP alloplasts with autografts, allografts and xenografts. MATERIALS AND METHODS In the present study, 4 types of bone grafting materials including autogenous bone harvested with a bone mill, DFDBA (LifeNet, USA), a xenograft derived from bovine bone mineral (NBM, BioOss, Geistlich, Switzerland) and a novel synthetic biphasic calcium phosphate (BCP, Straumman, Switzerland) were implanted into intramuscular pouches of 24 rats and analysed histologically for their ability to form ectopic bone formation around grafting particles. A semi-quantitative osteoinductive score was used to quantify the osteoinductive ability of each bone graft. RESULTS The results from the present study reveal that (1) autogenous bone resorbed rapidly in vivo, (2) the xenograft showed no potential to form ectopic bone formation and (3) both DFDBA and BCP were able to stimulate ectopic bone formation. CONCLUSION These studies demonstrate that these newly developed synthetic bone grafts have potential for inducing ectopic bone formation similar to DFDBA. Future clinical testing is necessary to reveal their bone-inducing properties in clinical scenarios including GBR procedures and in combination with implant dentistry. CLINICAL RELEVANCE Novel BCP scaffolds are able to induce ectopic bone formation without the use of osteoinductive growth factors such as BMP2 and thus demonstrate a large clinical possibility to further enhance bone formation for a variety of clinical procedures.

BACGROUND On June 5th, 2015 at Europerio 8, a group of leading experts were gathered to discuss what has now been 20 years of documented evidence supporting the clinical use of enamel matrix derivative (EMD). Original experiments led by Lars Hammarstrom demonstrated that enamel matrix proteins could serve as key regenerative proteins capable of promoting periodontal regeneration including new cementum, with functionally oriented inserting new periodontal ligament fibres, and new alveolar bone formation. This pioneering work and vision by Lars Hammarstrom has paved the way to an enormous amount of publications related to its biological basis and clinical use. Twenty years later, it is clear that all these studies have greatly contributed to our understanding of how biologics can act as mediators for periodontal regeneration and have provided additional clinical means to support tissue regeneration of the periodontium. AIMS This review article aims to: (1) provide the biological background necessary to understand the rational for the use of EMD for periodontal regeneration, (2) present animal and human histological evidence of periodontal regeneration following EMD application, (3) provide clinically relevant indications for the use of EMD and (4) discuss future avenues of research including key early findings leading to the development of Osteogain, a new carrier system for EMD specifically developed with better protein adsorption to bone grafting materials.

Gene therapy has garnished tremendous awareness for the repair of osseous defects. It exhibits high efficiency gene transfer and osteogenic differentiation potential making it well suitable for the sustained delivery of growth factors to local tissues. In the present study a simplified solution-based in situ biomimetic synthesis method is demonstrated for bone morphogenetic protein 7 (BMP7) adenovirus combined with silk fibroin scaffolds. This scaffold not only provides the three dimensional space for bone ingrowth, but also releases the BMP7 adenovirus which targets its secretion by host cells in vivo. Scaffolds were tested both in vitro for their osteogenic potential as well as in vivo in a critical-size calvarial defect in mice. Scaffolds loaded with bone morphogenetic protein 7 adenovirus (adBMP7) were able to sustain release of adBMP7 for up to 21 days and support cell proliferation and differentiation to bone forming osteoblasts. Calvarial defects treated with scaffolds containing adBMP7 significantly induced new bone formation in vivo. To demonstrate immuno-compatibility with host tissues, IL-2, IL-6 and TNF-α were measured up to 4 weeks post-implantation. Although these scaffolds demonstrated an initial pro-inflammatory response, levels of IL-2, IL-6 and TNF-α returned to baseline control values at either 2 or 4 weeks post-implantation demonstrating long term compatibility for growth factor delivery via gene therapy. The results from the present study indicate the promise of gene delivery scaffold systems for robust, low cost, and high quality bone tissue engineering applications.

Background: The clinical use of an enamel matrix derivative (EMD) has been shown to promote formation of new cementum, periodontal ligament (PDL), and bone and to significantly enhance the clinical outcomes after regenerative periodontal surgery. It is currently unknown to what extent the bleeding during periodontal surgery may compete with EMD adsorption to root surfaces. The aim of this study is to evaluate the effect of blood interactions on EMD adsorption to root surfaces mimicking various clinical settings and to test their ability to influence human PDL cell attachment and proliferation. Methods: Teeth extracted for orthodontic reasons were subjected to ex vivo scaling and root planing and treated with 24% EDTA, EMD, and/or human blood in six clinically related settings to determine the ability of EMD to adsorb to root surfaces. Surfaces were analyzed for protein adsorption via scanning electron microscopy and immunohistochemical staining with an anti-EMD antibody. Primary human PDL cells were seeded on root surfaces and quantified for cell attachment and cell proliferation. Results: Plasma proteins from blood samples altered the ability of EMD to adsorb to root surfaces on human teeth. Samples coated with EMD lacking blood demonstrated a consistent even layer of EMD adsorption to the root surface. In vitro experiments with PDL cells demonstrated improved cell attachment and proliferation in all samples coated with EMD (irrespective of EDTA) when compared to samples containing human blood. Conclusion: Based on these findings, it is advised to minimize blood interactions during periodontal surgeries to allow better adsorption of EMD to root surfaces.

Background: Autogenous bone grafts obtained by different harvesting techniques behave differently during the process of graft consolidation; the underlying reasons are however not fully understood. One theory is that harvesting techniques have an impact on the number and activity of the transplanted cells which contribute to the process of graft consolidation. Materials and Methods: To test this assumption, porcine bone grafts were harvested with four different surgical procedures: bone mill, piezosurgery, bone drilling (bone slurry), and bone scraper. After determining cell viability, the release of molecules affecting bone formation and resorption was assessed by reverse transcription polymerase chain reaction and immunoassay. The mitogenic and osteogenic activity of the conditioned media was evaluated in a bioassay with isolated bone cells. Results: Cell viability and the release of molecules affecting bone formation were higher in samples harvested by bone mill and bone scraper when compared with samples prepared by bone drilling and piezosurgery. The harvesting procedure also affected gene expression, for example, bone mill and bone scraper samples revealed significantly higher expression of growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor compared with the two other modalities. Receptor activator of nuclear factor kappa B ligand expression was lowest in bone scraper samples. Conclusion: These data can provide a scientific basis to better understand the impact of harvesting techniques on the number and activity of transplanted cells, which might contribute to the therapeutic outcome of the augmentation procedure.

OBJECTIVE Collagen membranes serve as barriers to separate bone grafts from soft tissues. Bone grafts harvested with a bone scraper release growth factors activating transforming growth factor-β (TGF-β) signaling in mesenchymal cells. The aim of the present pilot study was to determine whether collagen membranes adsorb molecules from bone-conditioned medium (BCM) with the capacity to provoke the expression of TGF-β target genes in vitro. MATERIALS AND METHODS Collagen membranes were soaked in aqueous extracts from fresh and demineralized bone chips placed in cell culture medium. Recombinant human TGF-β1 served as control. Gingival fibroblasts were seeded onto the washed collagen membranes and evaluated for the expression of adrenomedullin, pentraxin 3, interleukin 11, and proteoglycan 4. Cell viability and morphology with phalloidin staining were also determined. RESULTS Incubation of collagen membranes with BCM for at least one minute caused fibroblasts to decrease the expression of adrenomedullin and pentraxin 3, and to increase the expression of interleukin 11 and proteoglycan 4. Four different membrane treatments - incubated with recombinant TGF-β1, pre-wetted with saline solution, exposed to UV light, and dry out and stored one week at room temperature - also provoked significant changes in gene expression. Likewise, conditioned medium from demineralized bone chips caused gene expression changes. BCM did not alter the viability or morphology of gingival fibroblasts. CONCLUSIONS These findings demonstrate that collagen membranes rapidly adsorb the TGF-β activity released from bone chips, a molecular process that might contribute to guided bone regeneration.

BACKGROUND Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken. METHODS The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2. RESULTS The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days. CONCLUSION The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.

Introduction: The goal of regenerative periodontal therapy is the reconstitution of lost periodontal structures (i.e., the new formation of root cementum, periodontal ligament, and alveolar bone). Results from preclinical and clinical research in the last decade have provided evidence on the biologic rationale and clinical applications of an enamel matrix derivative (EMD) protein in periodontal wound healing and regeneration.Case Presentations and Literature Overview: This paper will provide an overview of the biologic rationale for using enamel matrix proteins (EMPs) in regenerative periodontal therapy. Based on the available preclinical and clinical evidence, the main clinical indications for using EMD in regenerative periodontal therapy will be discussed.Conclusions: The available data provide evidence of the biologic rationale of EMPs to support periodontal wound healing and regeneration. The application of EMD in conjunction with a surgical access may result in substantial regeneration of root cement...

Pathogenic bacterial infections and drug resistance make it urgent to develop new antibacterial agents with targeted delivery. Here, a new targeting delivery nanosystem is designed based on the potential interaction between bacterial recognizing receptors on macrophage membranes and distinct pathogen-associated molecular patterns in bacteria. Interestingly, the expression of recognizing receptors on macrophage membranes increases significantly when cultured with specific bacteria. Therefore, by coating pretreated macrophage membrane onto the surface of a gold-silver nanocage (GSNC), the nanosystem targets bacteria more efficiently. Previously, it has been shown that GSNC alone can serve as an effective antibacterial agent owing to its photothermal effect under near-infrared (NIR) laser irradiation. Furthermore, the nanocage can be utilized as a delivery vehicle for antibacterial drugs since the gold-silver nanocage presents a hollow interior and porous wall structure. With significantly improved bacterial adherence, the Sa-M-GSNC nanosystem, developed within this study, is effectively delivered and retained at the infection site both via local or systemic injections; the system also shows greatly prolonged blood circulation time and excellent biocompatibility. The present work described here is the first to utilize bacterial pretreated macrophage membrane receptors in a nanosystem to achieve specific bacterial-targeted delivery, and provides inspiration for future therapy based on this concept.

Leukocyte and platelet-rich fibrin (PRF), a second-generation platelet concentrate has been the focus of intensive research endeavors over the last 2 decades. Over the years, numerous reports have however failed to accurately report g-force values which have caused considerable confusion in the field. These values have since been re-transcribed incorrectly in many studies moving forward, and this article aims to address this topic to avoid further confusion in the field. We address several reports in which PRF centrifugal g-forces have been calculated at the PRF clot (referred to as relative centrifugal force [RCF]-clot) as opposed to the international standard method described at the bottom of centrifugation tubes (RCF-max). We further highlight how RCF-clot is not only a deviation from the standard international method used to report g-force values, but one subject to significant error owing to centrifugation time, patient hematocrit levels, initial volume of blood collected, and other factors. For these reasons and those further reported throughout this article, we address this controversy in detail to avoid further confusion regarding the report of g-force values in future studies. Furthermore, we propose a standardization regarding the accurate report of g-force values in future studies investigating PRF at the RCF-max.

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 1 h 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.

Bone morphogenetic protein-2 (BMP2) has been successfully utilized in dentistry to promote new bone formation because of its osteoinductive ability to recruit mesenchymal progenitor cells and induce their differentiation to bone-forming osteoblasts. Recently, novel biphasic calcium phosphate scaffolds have been developed with similar osteoinductive properties capable of forming ectopic bone formation.The aim of the present study was to assess whether the combination of BMP2 with this novel Biphasic Calcium Phosphate (BCP) scaffold may additionally promote new bone regeneration.Cylindrical bone defects measuring 2.5 mm were created bilaterally in the femurs of 18 Wistar rats. After 4 weeks, the following six groups were assessed for new bone formation by micro-computed tomography (CT) as well as histological assessment: 1) collagen scaffolds + 20 μg of BMP2; 2) collagen scaffolds + 50 μg of BMP2; 3) collagen scaffolds + 100 μg of BMP2; 4) BCP scaffolds + 20 μg of BMP2; 5) BCP scaffolds + 50 μg of BMP2; and 6) BCP scaffolds + 100 μg of BMP2. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining was utilized to assess osteoclast activity and osteoclast number. The release kinetics of BMP2 from both BCP and collagen scaffolds was investigated over a 14-day period.The results from present study demonstrate that BMP2 is able to promote new bone formation in a concentration dependant manner when loaded with either a collagen scaffolds or BCP scaffolds. Micro-CT analysis demonstrated significantly higher levels of new bone formation in groups containing BCP + BMP2 when compared with collagen scaffolds + BMP2. BMP2 had little effect on osteoclast activity; however, less TRAP staining and osteoclast number was observed in the defects receiving collagen scaffolds when compared with BCP scaffolds. The release of BMP2 over time was rapidly released after 1 day on BCP scaffolds whereas a gradually release over time was observed for collagen scaffolds up to 14 days.The osteoinductive properties of BMP2 may further be enhanced by its combination with a novel synthetically fabricated osteoinductive BCP scaffold. Future clinical testing is required to further assess these preliminary findings.

OBJECTIVE The aim of this study was to investigate the ability of Enamel Matrix Derivative (EMD) on vertical bone regeneration around dental implants placed in an extra-oral rabbit model. MATERIAL AND METHODS A total of 30 Straumann BL implants were partially embedded in transverse orientation into the posterior mandibles of 15 rabbits. Macro-structuring BiPhasicCaPST (BCPT1), micro-structuring BiPhasicCaPST (BCPT2), and deproteinized bovine bone mineral (DBBM) were placed around the implant and covered with a scaffold stabilizing "umbrella." EMD was incorporated within the scaffold for test sites, but not control sites. Histological analysis was performed on retrieved specimens after 10 weeks of healing to assess new bone formation. RESULTS All treatment groups displayed new supracrestal bone formation as determined by histomorphometric measurements, with mean values of new bone height ranging between 0.62 and 1.13 mm. Histological analysis revealed a higher mean bone formation (%) around the test sites where EMD (34.7, 95%CI: 27.1-39.4) was released from the scaffold, whereas the control group without EMD release (26.4, 95%CI: 16.3-31.9) (P = 0.069). The mean fBIC (%) in the BCPT2 group increased by the addition of EMD relative to no EMD (67.2, 95%CI: 48.6-84.1) and (54.7, 95%CI: 32.3-68.9), respectively). The BCPT2/EMD and DBBM/EMD interventions showed the greatest mean bone density (BA/TA), respectively, (12.8, 95%CI: 8.9-36.5) and (11.2, 6.3-16.4) in ROI 1. Values in ROI 2 were, similarly, (24.9, 95%CI: 17.2-31.7) and (27.7, 19.2-35.3). BA/TA in ROI 2 differences between the BCPT2 groups with and without EMD was statistically significant (P = 0.026), as well as the DBBM groups with and without EMD (P = 0.038). CONCLUSIONS A layer of new bone was formed in both test and controls. The release of EMD from BCPT2 and DBBM adjacent to a bone-level implant with an SLActive surface and scaffold retention umbrella consistently regenerated the greater fBIC and bone density along the length of the implant.

OBJECTIVES Bone replacement grafting materials play an important role in regenerative dentistry. Despite a large array of tested bone-grafting materials, little information is available comparing the effects of bone graft density on in vitro cell behavior. Therefore, the aim of the present study is to compare the effects of cells seeded on bone grafts at low and high density in vitro for osteoblast adhesion, proliferation, and differentiation. MATERIALS AND METHODS The response of osteoblasts to the presence of a growth factor (enamel matrix derivative, (EMD)) in combination with low (8 mg per well) or high (100 mg per well) bone grafts (BG; natural bone mineral, Bio-Oss®) density, was studied and compared for osteoblast cell adhesion, proliferation, and differentiation as assessed by real-time PCR. Standard tissue culture plastic was used as a control with and without EMD. RESULTS The present study demonstrates that in vitro testing of bone-grafting materials is largely influenced by bone graft seeding density. Osteoblast adhesion was up to 50 % lower when cells were seeded on high-density BG when compared to low-density BG and control tissue culture plastic. Furthermore, proliferation was affected in a similar manner whereby cell proliferation on high-density BG (100 mg/well) was significantly increased when compared to that on low-density BG (8 mg/well). In contrast, cell differentiation was significantly increased on high-density BG as assessed by real-time PCR for markers collagen 1 (Col 1), alkaline phosphatase (ALP), and osteocalcin (OC) as well as alizarin red staining. The effects of EMD on osteoblast adhesion, proliferation, and differentiation further demonstrated that the bone graft seeding density largely controls in vitro results. EMD significantly increased cell attachment only on high-density BG, whereas EMD was able to further stimulate cell proliferation and differentiation of osteoblasts on control culture plastic and low-density BG when compared to high-density BG. CONCLUSION The results from the present study demonstrate that the in vitro conditions largely influence cell behavior of osteoblasts seeded on bone grafts and in vitro testing. CLINICAL RELEVANCE These results also illustrate the necessity for careful selection of bone graft seeding density to optimize in vitro testing and provide the clinician with a more accurate description of the osteopromotive potential of bone grafts.

The health of peri-implant soft tissues is one of the most important aspects of osseointegration necessary for the long-term survival of dental implants.To review the process of soft tissue healing around osseointegrated implants and discuss the maintenance requirements as well as the possible short-comings of peri-implant soft tissue integration.Literature search on the process involved in osseointegration, soft tissue healing and currently available treatment modalities was performed and a brief description of each process was provided.The peri-implant interface has been shown to be less effective than natural teeth in resisting bacterial invasion because gingival fiber alignment and reduced vascular supply make it more vulnerable to subsequent peri-implant disease and future bone loss around implants. And we summarized common procedures which have been shown to be effective in preventing peri-implantitis disease progression as well as clinical techniques utilized to regenerate soft tissues with bone loss in advanced cases of peri-implantitis.Due to the difference between peri-implant interface and natural teeth, clinicians and patients should pay more attention in the maintenance and recovery of soft tissues around implants.

OBJECTIVES Enamel matrix derivative (EMD) has been successfully used for the regeneration of periodontal tissues including new cementum, periodontal ligament, and alveolar bone. Combination of EMD with bone grafting materials has however generated variable clinical results. Recently, we have demonstrated that a new formulation of EMD in a liquid carrier system (Osteogain®) has improved physicochemical properties for the adsorption of EMD to a bone grafting material. The aim of the present study was to investigate the regenerative potential of Osteogain®, in combination with a bone graft, on new bone formation in a rat femur defect model. MATERIALS AND METHODS Fifty-four critically sized femur defects (3 mm in diameter) were created bilaterally in 27 rats and treated following the group allocation: (1) drilled unfilled control, (2) a natural bone mineral (NBM), and (3) NBM + Osteogain®. All defects were histologically analyzed at 2, 4, and 8 weeks after surgical intervention. Micro-CT analysis, hematoxylin and eosin (H&E) staining, and Safranin O staining were performed to quantify new bone formation. RESULTS Significantly more new bone formation was observed in defects treated with NBM + Osteogain® at both 4 and 8 weeks when compared to NBM alone and the control unfilled defects (P

BACKGROUND The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

OBJECTIVES Osteoporosis is a disease characterized by a reduction in bone mass, poor bone strength, and microarchitectural deterioration primarily in postmenopausal women. With respect to periodontal disease, osteoporosis is thought to contribute to pre-existing alveolar degeneration although the association between both diseases is not fully characterized. The aim of the present study was to observe the initial changes in mandibular alveolar bone for sham-operated and ovariectomized (OVX) rats in ligature-induced experimental periodontitis. MATERIALS AND METHODS A total of 64 Wistar rats (7 weeks of age, 180-200 g) were used in this study (32 control sham-operated animals + ligature placement, 32 OVX animals + ligature placement). Following an 8-week period to induce an OVX model, micro-CT analysis was performed to calculate vertical and furcation bone loss of mandibular first molars at time points 0, 3, 7, and 11 days following ligature placement (six animals per group per time point). Furthermore, histological analysis was performed to calculate the loss of alveolar bone crest height from the cemento-enamel junction, and tartrate-resistant acid phosphatase (TRAP) staining was utilized to calculate the number of osteoclasts. RESULTS The results from the present study demonstrate that OVX animals showed significant vertical bone loss at all time points when compared to control sham-operated animals. In the furcation area, no significant difference in bone loss was observed between sham-operated and OVX animals at 0, 3, and 7 days; however by 11 days, a significant decrease in bone volume/total volume and trabecular thickness was observed in the OVX group. The histological analysis also revealed that alveolar bone crest height was significantly reduced in OVX animals, and TRAP staining further revealed the greater number of multinucleated osteoclasts peaking at 3 days postligature placement. CONCLUSION The results from the present study demonstrate a direct correlation between the osteoporotic phenotype and the progression of periodontal breakdown in a diseased-induced animal model. CLINICAL RELEVANCE It may be suggested that an osteoporotic phenotype has the potential to speed periodontal breakdown and thus contributes to the overall degeneration of the periodontium in patients suffering from postmenopausal bone loss. Future research from human clinical studies are necessary to further understand the relationship between periodontal disease and osteoporosis.

Over 15 years have passed since an enamel matrix derivative (EMD) was introduced as a biologic agent capable of periodontal regeneration. Histologic and controlled clinical studies have provided evidence for periodontal regeneration and substantial clinical improvements following its use. The purpose of this review article was to perform a systematic review comparing the eff ect of EMD when used alone or in combination with various types of bone grafting material.A literature search was conducted on several medical databases including Medline, EMBASE, LILACS, and CENTRAL. For study inclusion, all studies that used EMD in combination with a bone graft were included. In the initial search, a total of 820 articles were found, 71 of which were selected for this review article. Studies were divided into in vitro, in vivo, and clinical studies. The clinical studies were subdivided into four subgroups to determine the eff ect of EMD in combination with autogenous bone, allografts, xenografts, and alloplasts.The analysis from the present study demonstrates that while EMD in combination with certain bone grafts is able to improve the regeneration of periodontal intrabony and furcation defects, direct evidence supporting the combination approach is still missing.Further controlled clinical trials are required to explain the large variability that exists amongst the conducted studies.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Replacement bone grafting materials are used clinically for a variety of clinical procedures to augment and replace lost or missing bone. Little information is available regarding their degradation properties.The aim of the present study was to investigate the degradation rate and modes of degradation of four commonly used bone grafting materials.A natural bone mineral (NBM) of bovine origin, NBM in combination with enamel matrix derivative (EMD), LifeNet demineralized freeze-dried bone allograft (DFDBA), and Osteotech DFDBA were analyzed for particle degradation over time in 3 mm femur defects created in female Wistar rats. At 2, 4, and 8 weeks postimplantation, femur defects were assigned to histological analysis. Hematoxylin and eosin, Safranin O, tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa B ligand (RANKL), and matrix metalloproteinase-2 (MMP-2) staining were performed to determine the rate of particle degradation, number of osteoclasts around particles, and intensity and localization of TRAP, RANKL, and MMP-2 staining.In the present study, NBM particles demonstrated little signs of degradation. The combination of NBM with EMD significantly increased the number of osteoclasts around NBM particles and increased expression of RANKL and MMP-2 specifically around particle surface. Only minor resorption was observed. Both DFDBA particles showed much faster degradation of particles. Interestingly, fewer osteoclasts were found on their surface when compared with NBM particles, specifically on Osteotech DFDBA particles, suggesting an alternative mode of degradation. Osteotech DFDBA particles demonstrated significantly faster degradation when compared with all other bone grafts. No obvious increase in TRAP, RANKL, or MMP2 was observed to validate this fast rate of degradation.The results from the present study demonstrate a wide range of particle degradation between various commonly commercially available bone grafts. Further research to determine the precise mechanisms that influence particle degradation is necessary.

New development of biomaterial scaffolds remains a prominent issue for the regeneration of lost or fractured bone. Of these scaffolds, a number of bioactive polymers have been synthesized and fabricated for diverse biological roles. Although recent evidence has demonstrated that composite scaffolds such as HA/PLLA have improved properties when compared to either HA or PLLA alone, recent investigations have demonstrated that the phase compatibility between HA and PLLA layers is weak preventing optimal enhancement of the mechanical properties and making the composites prone to breakdown. In the present study, poly (γ-benzyl-L-glutamate) modified hydroxyapatite/(poly (L-lactic acid)) (PBLG-g-HA/PLLA) composite scaffolds were fabricated with improved phase compatibility and tested for their osteogenic properties in 18 Wistar female rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. At time points, 2, 4 and 8 weeks post surgery, bone formation was evaluated by µ-CT and histological analysis by comparing 4 treatment groups; 1) blank defect, 2) PLLA, 3) HA/PLLA and 4) PBLG-g-HA/PLLA scaffolds. The in vivo analysis demonstrated that new bone formation was much more prominent in HA/PLLA and PBLG-g-HA/PLLA groups as depicted by µ-CT, H&E staining and immunohistochemistry for collagen I. TRAP staining was also utilized to determine the influence of osteoclast cell number and staining intensity to the various scaffolds. No significant differences in either staining intensity or osteoclast numbers between all treatment modalities was observed, however blank defects did contain a higher number of osteoclast-like cells. The results from the present study illustrate the potential of PBLG-g-HA/PLLA scaffolds for bone tissue engineering applications by demonstrating favorable osteogenic properties.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p