Impact of Freeze-dried Corticocancellous Bone Allograft Combined with Enamel Matrix Derivative in the Treatment of Critical-sized Calvarial Bone Defects: An Animal Study.

AIM: This study compared the quality and quantity of newly formed bone in rabbits' critical-sized calvarial defects filled with enamel matrix derivative (EMD) combined with freeze-dried bone allograft (FDBA) vs FDBA alone. MATERIALS AND METHODS: A total of 24 adult male white New Zealand rabbits were included. In each rabbit, three bone defects with a diameter of 8 mm were created on the calvarium bone; the first defect was left untreated, while the second was filled with FDBA, and the third was filled with EMD + FDBA. Twelve rabbits were randomly euthanized after a month, and the remaining 2 month postsurgery. Bone sections were histologically evaluated by hematoxylin and eosin and vascular endothelial growth factor (VEGF), alkaline phosphatase (ALP), osteoprotegerin (OPG), and receptor activator of NF-kappaB (RANK) immune-histochemical staining. RESULTS: An improvement in the newly formed bone percentage was found in the defects filled with EMD + FDBA in comparison with FDBA and control defects at 1 month and 2 months postsurgery. Additionally, the expression of VEGF, ALP, OPG, and RANK showed highly significant differences in the defects filled with EMD + FDBA compared to the FDBA and control ones at 1 month postsurgery (p = 0.001). Meanwhile, VEGF and ALP expression showed a significant decrease in defects filled with EMD + FDBA compared to the FDBA and control ones (p = 0.001), while OPG and RANK expression showed non-significant differences between treated groups at 2 months postsurgery. CONCLUSION: Enamel matrix derivative combined with FDBA has a synergistic effect on bone formation and graft substitution. This combination accelerates the expression of VEGF, ALP, OPG, and RANK. CLINICAL SIGNIFICANCE: The combination of EMD and FDBA accelerates and ameliorates the quality of newly formed bone, aiding in maxillofacial reconstruction. How to cite this article: Zakri RN, Grawish ME, Mowafey B, et al. Impact of Freeze-dried Corticocancellous Bone Allograft Combined with Enamel Matrix Derivative in the Treatment of Critical-sized Calvarial Bone Defects: An Animal Study. J Contemp Dent Pract 2024;25(5):424-431.

EMD-liquid contributes to tissue thickness gain in soft tissue augmentation using a collagen matrix.

OBJECTIVES: To investigate the function of enamel matrix derivative (EMD)-liquid compared to EMD-gel (original Emdogain® with polyglycolic acid-carrier) in inducing soft tissue regeneration using a rat dorsal model. MATERIAL AND METHODS: Four subcutaneous pouches were created through dorsal skin incisions in 18 female Wistar rats and randomly allocated to the following groups: (1) sterile saline + non-crosslinked collagen matrix (CM), (2) EMD-gel + CM, and (3) EMD-liquid + CM. After 2 and 4 weeks of healing, the specimens were harvested and stained with Goldner's trichrome, hematoxylin and eosin, and were immunohistochemically stained with an anti-CD31 antibody. RESULTS: The EMD-liquid group showed the thickest connective tissue compared to the other groups, with statistical significance both at 2 (p < 0.001) and 4 weeks (p = 0.011 and 0.023, respectively). The number of multinucleated giant cells was not significantly different among the groups for both periods. Moreover, there was a tendency to have more blood vessels over a longer period, and the highest number of blood vessels was observed in the EMD-liquid group at 4 weeks (p = 0.009 and 0036, respectively). CONCLUSION: EMD-liquid-treated CM is advantageous compared to using CM alone or EMD-gel-treated CM, owing to the histomorphometric results that show significantly increased soft tissue thickness and number of blood vessels when EMD-liquid was pre-primed to CM. CLINICAL RELEVANCE: EMD with a liquid carrier may be an appropriate biologic supplement to provide cell-inducing properties to the CM scaffold and is clinically more beneficial for phenotype modification therapy than CM only and EMD-gel-treated CM.

Influence of Occlusal Hypofunction on Alveolar Bone Healing in Rats.

The aim of this in vivo study was to investigate the effect of occlusal hypofunction on alveolar bone healing in the absence or presence of an enamel matrix derivative (EMD). A standardized fenestration defect over the root of the mandibular first molar in 15 Wistar rats was created. Occlusal hypofunction was induced by extraction of the antagonist. Regenerative therapy was performed by applying EMD to the fenestration defect. The following three groups were established: (a) normal occlusion without EMD treatment, (b) occlusal hypofunction without EMD treatment, and (c) occlusal hypofunction with EMD treatment. After four weeks, all animals were sacrificed, and histological (hematoxylin and eosin, tartrate-resistant acid phosphatase) as well as immunohistochemical analyses (periostin, osteopontin, osteocalcin) were performed. The occlusal hypofunction group showed delayed bone regeneration compared to the group with normal occlusion. The application of EMD could partially, but not completely, compensate for the inhibitory effects of occlusal hypofunction on bone healing, as evidenced by hematoxylin and eosin and immunohistochemistry for the aforementioned molecules. Our results suggest that normal occlusal loading, but not occlusal hypofunction, is beneficial to alveolar bone healing. Adequate occlusal loading appears to be as advantageous for alveolar bone healing as the regenerative potential of EMD.

Comparison of the effectiveness of Ankaferd Blood Stopper® and Emdogain in periodontal regeneration.

OBJECTIVES: The present study was performed to compare the effectiveness of Ankaferd Blood Stopper® (ABS) with enamel matrix derivatives (EMD) for treating fenestration defects in rats. MATERIALS AND METHODS: Forty-eight male Wistar rats were randomly divided into six groups (each n = 8). Fenestration defects were created in all rats, to which ABS, EMD, or saline (S) was then applied. The rats were grouped and sacrificed at one of two different time points, as follows: ABS-10-group, ABS-treatment/sacrifice on day 10; EMD-10-group, EMD-treatment/sacrifice on day 10; S-10-group, S-treatment/sacrifice on day 10; ABS-38-group, ABS-treatment/sacrifice on day 38; EMD-38-group, EMD-treatment/sacrifice on day 38; and S-38-group, S-treatment/sacrifice on day 38. Then, histomorphometric analysis including measurements of new bone area (NBA) and new bone ratio (NBR), and immunohistochemical analysis including the determination of osteopontin (OPN) and type-III-collagen (C-III) expression were performed. RESULTS: The NBA and NBR were significantly higher in the ABS-10-group and EMD-10-group compared to the S-10-group (p < .05), and in the EMD-38-group compared to the S-38-group (p < .05). The levels of C-III and OPN immunoreactivity were significantly higher in the ABS-10-group compared to the S-10-group (p < .017). CONCLUSIONS: The results of this study suggested that ABS can promote early periodontal regeneration, although its efficacy seems to decrease over time.

Apical approach in periodontal reconstructive surgery with enamel matrix derivate and enamel matrix derivate plus bone substitutes: a randomized, controlled clinical trial.

OBJECTIVES: This parallel, randomized controlled clinical trial evaluated the influence of bone substitutes (BS) on the efficacy of the non-incised papillae surgical approach (NIPSA) with enamel matrix derivate (EMD) in resolving deep, isolated, combined non-contained intrabony and supra-alveolar periodontal defects, preserving the soft tissue. MATERIAL AND METHODS: Twenty-four patients were randomized to treatment with NIPSA and EMD or NIPSA plus EMD and BS. Bleeding on probing (BoP), interproximal clinical attachment level (CAL), interproximal probing depth (PD), recession (REC), location of the tip of the papilla (TP), and width of the keratinized tissue (KT) were evaluated before surgery and at 1 year post-surgery (primary outcomes). Wound closure was assessed at 1 week post-surgery, and supra-alveolar attachment gain (SUPRA-AG) was recorded at 1 year post-surgery. RESULTS: At 1 week, 87.5% of cases registered complete wound closure and there were no cases of necrosis, without differences between groups (p > .05). At 1 year, all cases showed negative BoP. A significant PD reduction (NIPSA + EMD 8.25 ± 2.70 mm vs. NIPSA + EMD + BS 6.83 ± 0.81 mm) and CAL gain (NIPSA + EMD 8.33 ± 2.74 mm vs. NIPSA + EMD + BS 7.08 ± 2.68 mm) were observed (p < .001) in both groups, without significant between-group differences (p > .05). The residual PD was < 5 mm in all defects (NIPSA + EMD 2.50 ± 0.67 mm vs. NIPSA + EMD + BS 2.67 ± 0.78 mm). Soft tissues were preserved without significant between-group differences (REC: NIPSA + EMD 0.25 ± 0.45 mm vs. NIPSA + EMD + BS 0.17 ± 0.58 mm, p > .05; KT: 0.00 ± 0.43 mm vs. 0.08 ± 0.67 mm, p > .05). There were improvements in the papilla in both groups (TP: NIPSA + EMD 0.33 ± 0.49 mm vs. NIPSA + EMD + BS 0.45 ± 0.52 mm, p > .05), which was only significant in the NIPSA EMD + BS group (0.45 ± 0.52 mm; p < .05). In both groups, CAL gain was recorded in the supra-alveolar component, showing full resolution of the intrabony component of the defect in all cases (SUPRA-AG: NIPSA + EMD 1.83 ± 1.11 mm vs. NIPSA + EMD + BS 2.00 ± 1.76 mm, p > .05). CONCLUSIONS: NIPSA and EMD with or without BS seem to be a valid surgical approach in the treatment of isolated, deep non-contained periodontal defects. In our study, both treatments resulted in significant PD reduction and CAL gain, that extended in the supra-alveolar component, without differences with the use of BS. Both treatments resulted in soft tissue preservation. However, the addition of BS may improve interdental papillary tissue. CLINICAL RELEVANCE: NIPSA, with or without bone substitutes, resulted in significant periodontal improvement, with soft tissue preservation in isolated, deep non-contained periodontal defects. The application of bone substitutes may provide interproximal soft tissue gain. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov: NCT04712630.

TGF-ß Signalling Mediates the Anti-Inflammatory Activity of Enamel Matrix Derivative In Vitro.

Enamel matrix derivative (EMD) prepared from extracted porcine fetal tooth material can support the regrow of periodontal tissues. Previous findings suggest that EMD has anti-inflammatory properties and TGF-ß activity in vitro. However, the anti-inflammatory activity of EMD is mediated via TGF-ß has not been considered. To this aim, we first established a bioassay to confirm the anti-inflammatory activity of EMD. The bioassay was based on the RAW 264.7 macrophage cell line and proven with primary macrophages where EMD significantly reduced the forced expression of IL-6. We then confirmed the presence of TGF-ß1 in EMD by immunoassay and by provoking the Smad2/3 nuclear translocation in RAW 264.7 macrophages. Next, we took advantage of the TGF-ß receptor type I kinase-inhibitor SB431542 to block the respective signalling pathway. SB431542 reversed the anti-inflammatory activity of EMD and TGF-ß in a bioassay when IL-6 and CXCL2 expression was driven by the LPS stimulation of RAW 264.7 macrophages. This central observation was supported by showing that SB431542 reversed the anti-inflammatory activity of EMD using IL-1ß and TNF-α-stimulated ST2 bone marrow stromal cells. Together, these findings implicate that the TGF-ß activity mediates at least part of the anti-inflammatory activity of EMD in vitro.

Enamel Matrix Derivative Decreases Pyroptosis-Related Genes in Macrophages.

Background: Pyroptosis is a caspase-dependent catabolic process relevant to periodontal disorders for which inflammation is central to the pathophysiology of the disease. Although enamel matrix derivative (EMD) has been applied to support periodontal regeneration, its capacity to modulate the expression of pyroptosis-related genes remains unknown. Considering EMD has anti-inflammatory properties and pyroptosis is linked to the activation of the inflammasome in chronic periodontitis, the question arises whether EMD could reduce pyroptosis signalling. Methods: To answer this question, primary macrophages obtained from murine bone marrow and RAW 264.7 macrophages were primed with EMD before being challenged by lipopolysaccharide (LPS). Cells were then analysed for pyroptosis-signalling components by gene expression analyses, interleukin-1ß (IL-1ß) immunoassay, and the detection of caspase-1 (CAS1). The release of mitochondrial reactive oxygen species (ROS) was also detected. Results: We report here that EMD, like the inflammasome (NLRP3) and CAS1 specific inhibitors-MCC950 and Ac-YVAD-cmk, respectively-lowered the LPS-induced expression of NLRP3 in primary macrophages (EMD: p = 0.0232; MCC950: p = 0.0426; Ac-YVAD-cmk: p = 0.0317). EMD further reduced the LPS-induced expression of NLRP3 in RAW 264.7 cells (p = 0.0043). There was also a reduction in CAS1 and IL-1ß in RAW 264.7 macrophages on the transcriptional level (p = 0.0598; p = 0.0283; respectively), in IL-1ß protein release (p = 0.0313), and CAS1 activity. Consistently, EMD, like MCC950 and Ac-YVAD-cmk, diminished the ROS release in activated RAW 264.7 cells. In ST2 murine mesenchymal cells, EMD could not be tested because LPS, saliva, and IL-1ß + TNF-α failed to provoke pyroptosis signalling. Conclusion: These findings suggest that EMD is capable of dampening the expression of pyroptosis-related genes in macrophages.

Ameloblastin attenuates RANKL-mediated osteoclastogenesis by suppressing activation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1).

Ameloblastin (Ambn) is an extracellular matrix protein and member of the family of enamel-related gene products. Like amelogenin, Ambn is mainly associated with tooth development, especially biomineralization of enamel. Previous studies have shown reductions in the skeletal dimensions of Ambn-deficient mice, suggesting that the protein also has effects on the differentiation of osteoblasts and/or osteoclasts. However, the specific pathways used by Ambn to influence osteoclast differentiation have yet to be identified. In the present study, two cellular models, one based on bone marrow cells and another on RAW264.7 cells, were used to examine the effects of Ambn on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. The results showed that Ambn suppresses osteoclast differentiation, cytoskeletal organization, and osteoclast function by the downregulation of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, actin ring formation, and areas of pit resorption. The expression of the osteoclast-specific genes TRAP, MMP9, cathepsin K, and osteoclast stimulatory transmembrane protein (OC-STAMP) was abolished in the presence of Ambn, while that of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), the master regulatory factor of osteoclastogenesis, was also attenuated by the downregulation of c-Fos expression. In Ambn-induced RAW264.7 cells, phosphorylation of cAMP-response element-binding protein (CREB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was reduced. Calcium oscillation was also decreased in the presence of Ambn, suggesting its involvement in both RANKL-induced osteoclastogenesis and costimulatory signaling. B-lymphocyte-induced maturation protein-1 (Blimp1), a transcriptional repressor of negative regulators of osteoclastogenesis, was also downregulated by Ambn, resulting in the elevated expression of v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB), B-cell lymphoma 6 (Bcl6), and interferon regulatory factor-8 (Irf8). Taken together, these findings suggest that Ambn suppresses RANKL-induced osteoclastogenesis by modulating the NFATc1 axis.

Does enamel matrix derivative application improve clinical outcomes after semilunar flap surgery? A randomized clinical trial.

OBJECTIVES: To evaluate the treatment of gingival recessions by semilunar coronally positioned flap plus enamel matrix derivative (SCPF + EMD). MATERIALS AND METHODS: Thirty patients with class I localized gingival recession were included. They were randomly allocated in two groups: SCPF + EMD and SCPF. Recession height (RH), recession width (RW), width of keratinized tissue (WKT), thickness of keratinized tissue (TKT), probing depth (PD), and clinical attachment level (CAL) were measured at baseline, 6 and 12 months post-surgery. Patient/professional evaluation of esthetics and root sensitivity was performed. RESULTS: After 12 months, mean root coverage was 1.98 ± 0.33 mm for SCPF + EMD (90.86 ± 14.69%) and 1.85 ± 0.41 mm (79.76 ± 17.44%) for SCPF (p > 0.05). The esthetic evaluation by the patient showed preference for SCPF + EMD. According to the professional evaluation (QCE), the use of EMD decreases the appearance of postoperative scar tissue line. There was a significant reduction in root hypersensitivity with no further complaints by the patients. CONCLUSIONS: The addition of EMD provides significantly better esthetics to SCPF, according to patient and professional assessments. SCPF + EMD is effective but not superior to SCPF for root coverage, after 12 months. CLINICAL RELEVANCE: Previous clinical trials showed that the combination of EMD with coronally advanced flaps may enhance the outcome of root coverage. There is a lack of studies testing the combination of EMD with SCPF. The combination SCPF + EMD provides better esthetics when compared to the SCPF and is effective, but not superior, to SCPF for root coverage, after 12 months. TRIAL REGISTRATION: NCT02459704.

Cytokine expression in response to root repair agents.

AIM: To evaluate the expression of TNF-α, IL-6, IFN-γ, TGF-ß, IL-4, IL-10, RANKL, RANK and OPG on mouse calvarial bone treated with MTA, Geristore® and Emdogain® . METHODOLOGY: Bone wounds were made on the heads of C57BL/6 mice, breaking the periosteum and the cortical surface of the calvaria. Each repair agent was inserted into sectioned Eppendorf microtubes and placed on the bone wound, and soft tissues were sutured. At 14 and 21 days, animals were sacrificed and the treated region was dissected. The calvaria bone was removed, and RNA was extracted. mRNA expression of the aforementioned cytokines was assessed using real-time PCR. Data were analysed by nonparametric methods, including the Mann-Whitney and Kruskal-Wallis tests (P < 0.05). RESULTS: Following treatment with Emdogain® and MTA, mRNA expression of RANKL, RANK and OPG increased significantly (P < 0.05) between days 14 to 21. Geristore® did not alter the basal expression of these mediators during the same period of evaluation. Whilst treatment with Emdogain® did cause a significant increase in TNF-α mRNA expression between days 14 and 21 (P < 0.05), treatment with MTA did not alter the basal expression of this cytokine at either experimental time point. However, TNF-α mRNA expression was down-regulated significantly at day 21 (P < 0.05) when Geristore® was applied. A significant increase in the mRNA expression of IL-6, TGF-ß, IL-10, IL-4 and IFN-γ was observed with Emdogain® and MTA treatment between days 14 to 21, whereas Geristore® reduced significantly the expression of IL-6, TGF-ß and IL-4 (P < 0.05). CONCLUSION: The clinical indication of these repair agents depends on the root resorption diagnosis. Whilst MTA and Emdogain® induce a pro- and anti-inflammatory response early and late, respectively, Geristore® was not associated with an inflammatory reaction when compared with both repair agents.

Evaluation of Emdogain® antimicrobial effectiveness against biofilms containing the keystone pathogen Porphyromonas gingivalis.

This study aimed to evaluate the antimicrobial activity of Emdogain® (EMD) against biofilms containing the periopathogen Porphyromonas gingivalis. A brain-Heart infusion broth inoculated with S. gordonii and P. gingivalis was perfused (7-d, anaerobiosis) through a closed circuit containing two Robbins devices as to form biofilms. The latter were then treated for 2 min with various antimicrobials (Chlorhexidine (CHX) 0.2%, Povidone iodine (PVI) 5%, PVI 10%, essential oils (EO), EO ZeroTM or EMD) (n=8) and cell densities were calculated and compared. In the present in vitro model, Emdogain® was not statistically effective (p>0.05) in killing biofilm bacteria unlike the other tested molecules.

Biofunctionalization of porcine-derived collagen matrix using enamel matrix derivative and platelet-rich fibrin: influence on mature endothelial cell characteristics in vitro.

OBJECTIVES: The present study evaluated the effect of an enamel matrix derivative (EMD) and platelet-rich fibrin (PRF)-modified porcine-derived collagen matrix (PDCM) on human umbilical vein endothelial cells (HUVEC) in vitro. MATERIALS AND METHODS: PDCM (mucoderm®) was prepared to 6 mm (±0.1 mm) diameter discs. PDCM samples were incubated with either EMD, PRF, or control solutions for 100 min at 4 °C before the experiments. Cell-inducing properties of test materials on HUVEC cells were tested with cell proliferation assays (MTT, PrestoBlue®), a cytotoxicity assay (ToxiLight®), a Boyden chamber migration assay, and a cell attachment assay. Scanning electron microscopy (SEM) imaging was performed to determine the surface and the architecture of the modified matrices. RESULTS: Cell proliferation was elevated in the EMD and PRF groups compared with control (p each ≤0.046). PRF modification increased HUVEC migration ability by 8-fold compared with both control and EMD groups (p each <0.001). Both treatments significantly promoted the cell attachment of HUVEC to PDCM, as assessed by direct cell counts on the matrices (p each <0.001). CONCLUSIONS: HUVEC cell characteristics were overall improved by EMD- and PRF- modified PDCM. Adsorbed bioactive molecules to the PDCM surface may have contributed to a more preferable environment to surrounding cells. CLINICAL RELEVANCE: The results may give evidence that PDCM modification with EMD or PRF, respectively, might be a useful approach to improve clinical outcomes, to prevent inflammatory reactions and wound-healing disturbances, and to expand the clinical application area of PDCM.

Histological healing after nonsurgical periodontal treatment with enamel matrix derivatives in canine experimental periodontitis.

The histological outcomes after nonsurgical periodontal treatment with enamel matrix derivatives (EMD) remain controversial. The present study evaluated periodontal wound healing after scaling and root planing (SRP) with subgingival application of EMD for treatment of experimental periodontitis. Periodontal breakdown was induced by applying silk ligatures around mandibular third and fourth premolars of six beagle dogs until radiographic bone loss progressed to approximately half of the root length. Probing pocket depth (PPD) and clinical attachment level (CAL) were proximally measured 2 weeks after ligature removal (baseline). Mesial and distal surfaces of the experimental teeth were subjected to SRP and randomized using a split-mouth design to subgingival application of EMD (test) or normal saline (control). PPD and CAL were re-evaluated at 11 weeks. Animals were sacrificed at 12 weeks for histological analyses. No significant differences were observed in PPD and CAL between both groups at baseline and at 11 weeks. Histologically, test sites exhibited a greater amount of new cementum than that did the control sites (p < 0.01). Moreover, the control sites revealed increased epithelial downgrowth compared with the test sites: (p < 0.05). On the other hand, no intergroup differences were detected in terms of bone position, connective tissue attachment, gingival recession, and planed root length. This study suggested that EMD has an increased potential to support formation of new cementum with decreased epithelial downgrowth when used as an adjunct to nonsurgical periodontal treatment.

Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway.

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.

Osteogain improves osteoblast adhesion, proliferation and differentiation on a bovine-derived natural bone mineral.

BACKGROUND: The use of enamel matrix derivative (EMD) has been shown to facilitate periodontal regeneration by histologically resulting in formation of cementum, periodontal ligament and bone. Recently, a new liquid carrier system for EMD has been introduced with better physicochemical properties specifically designed for bone graft mixing (Osteogain). The aim of this study was to investigate the combination of Osteogain with a bovine-derived natural bone mineral (NBM) on osteoblast migration, adhesion, proliferation and differentiation. MATERIALS AND METHODS: Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto 1)NBM particles alone or 2)NBM + Osteogain. Samples were compared for cell migration at 8 h, cell adhesion at 4 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 3 and 14 days for genes encoding runt-related transcription factor 2 (Runx2), collagen1alpha2 (COL1a2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, alizarin red staining was utilized to investigate the mineralization at 14 days. RESULTS: Osteogain significantly upregulated cell adhesion over twofold onto NBM particles and promoted cell proliferation at 3 and 5 days after seeding. Furthermore, the combination of NBM with Osteogain significantly upregulated genes encoding Runx2, ALP, COL1a2 and OCN (from 1.5- to 3-fold) and increased alizarin red staining over 3 fold at 14 days when compared to NBM particles alone. CONCLUSION: Pre-coating Osteogain onto NBM bone grafting particles significantly increased cell adhesion, proliferation and differentiation of osteoblasts in vitro. Future animal studies are now necessary to further investigate the regenerative potential of Osteogain in combination with a bone grafting material prior to clinical use for bone regeneration.

A novel flapless approach versus minimally invasive surgery in periodontal regeneration with enamel matrix derivative proteins: a 24-month randomized controlled clinical trial.

OBJECTIVES: This investigation was designed to compare the effectiveness of enamel matrix derivative (EMD) proteins in combination with flapless or flap procedure in periodontal regeneration of deep intrabony defects. MATERIALS AND METHODS: Thirty chronic periodontitis patients who had at least one residual periodontal defect with an intrabony component of ≥3 mm were consecutively enrolled. Defects were randomly assigned to test or control treatments which both consisted of the use of EMD to reach periodontal regeneration. Test sites (n = 15) were treated according to a novel flapless approach, whereas control sites (n = 15) by means of minimally invasive surgery (MIST). Clinical and radiographic parameters were recorded at baseline, 12 and 24 months post-operatively. RESULTS: Both therapeutic modalities yielded similar probing depth (PD) reduction and clinical attachment level (CAL) gain at 24 months. In flapless-treated sites, a mean PD reduction of 3.6 ± 1.0 mm and a CAL gain of 3.2 ± 1.1 mm were observed. In the MIST group, they were 3.7 ± 0.6 and 3.6 ± 0.9 mm. The operative chair time was twice as long in the MIST compared to the flapless group, whereas comparable patient-oriented outcomes were observed. CONCLUSION: The flapless procedure may be successfully applied in the regenerative treatment of deep intrabony defects reaching clinical outcomes comparable with those of minimally invasive surgical approaches and may present important advantages in terms of reduction of operative chair time. CLINICAL RELEVANCE: The use of EMD as an adjunct to non-surgical periodontal treatment may be considered a suitable option to treat defects mainly in the anterior sextants.

Osteogain® loaded onto an absorbable collagen sponge induces attachment and osteoblast differentiation of ST2 cells in vitro.

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.

Differential effects of tyrosine-rich amelogenin peptide on chondrogenic and osteogenic differentiation of adult chondrocytes.

Current approaches to treat osteoarthritis (OA) are insufficient. Autologous chondrocyte implantation (ACI) has been used for the past decade to treat patients with OA or focal cartilage defects. However, a number of complications have been reported post-ACI, including athrofibrosis and symptomatic hypertrophy. Thus, a long-term ACI strategy should ideally incorporate methods to 'prime' autologous chondrocytes to form a cartilage-specific matrix and suppress hypertrophic mineralization. The objective of this study is to examine the effects of tyrosine-rich amelogenin peptide (TRAP; an isoform of the developmental protein amelogenin) on human articular cartilage cell (HAC) chondrogenic differentiation and hypertrophic mineralization in vitro. Effects of chemically synthesized TRAP on HAC chondrogenic differentiation were determined by assessing: (1) sGAG production; (2) Alcian blue staining for proteoglycans; (3) collagen type II immunostaining; and (4) expression of the chondrogenic genes SOX9, ACAN and COL2A1. Hypertrophic mineralization was assayed by: (1) ALP expression; (2) Alizarin red staining for Ca(+2)-rich bone nodules; (3) OC immunostaining; and (4) expression of the osteogenic/hypertrophic genes Ihh and BSP. Chemically synthesized TRAP was found to suppress terminal osteogenic differentiation of HACs cultured in hypertrophic mineralization-like conditions, an effect mediated via down-regulation of the Ihh gene. Moreover, TRAP was found to augment chondrogenic differentiation of HACs via induction of SOX9 gene expression when cells were cultured in pro-chondrogenic media. The results obtained from this proof-of-concept study motivate further studies on the use of TRAP as part of a preconditioning regimen in autologous chondrocyte implantation procedures for OA patients and patients suffering from focal cartilage defects.

Enamel matrix protein derivative and/or synthetic bone substitute for the treatment of mandibular class II buccal furcation defects. A 12-month randomized clinical trial.

OBJECTIVE: This study aims to clinically evaluate the treatment of mandibular class II furcation defects with enamel matrix derivative (EMD) and/or a bone substitute graft made of ß-tricalcium phosphate/hydroxyapatite (ßTCP/HA). MATERIALS AND METHODS: Forty-one patients, presenting a mandibular class II buccal furcation defect, probing pocket depth (PPD) ≥4 mm and bleeding on probing, were included. They were randomly assigned to the groups: 1-EMD (n = 13); 2-ßTCP/HA (n = 14); 3-EMD + ßTCP/HA (n = 14). Plaque index (PI), gingival index (GI), relative gingival margin position (RGMP), relative vertical and horizontal attachment level (RVCAL and RHCAL), and PPD were evaluated at baseline and 6 and 12 months. The mean horizontal clinical attachment level gain was considered the primary outcome variable. RESULTS: No significant intragroup differences were observed for RGMP, but significant changes were observed for RVCAL, RHCAL, and PPD for all groups (p < 0.05). After 12 months, the mean horizontal clinical attachment level gain was 2.77 ± 0.93 mm for EMD, 2.64 ± 0.93 mm for ßTCP/HA, and 2.93 ± 0.83 mm for EMD + ßTCP/HA, with no significant differences among the groups. At the end of the study, 85.3 % of the sites were partially closed; however, no complete closure was observed. CONCLUSION: EMD + ßTCP/HA does not provide a significant advantage when compared to the isolated approaches. All three tested treatments promote significant improvements and partial closure of class II buccal furcation defects. Based on its potential to induce periodontal regeneration, EMD may be considered an attractive option for this type of defect, but complete closure remains an unrealistic goal. CLINICAL RELEVANCE: The partial closure of buccal furcation defects can be achieved after the three tested approaches. However, the combined treatment does not provide a significant benefit when compared to the isolated approaches.

Healing of localized gingival recessions treated with a coronally advanced flap alone or combined with an enamel matrix derivative and a porcine acellular dermal matrix: a preclinical study.

OBJECTIVE: This study aimed to evaluate the effects of a porcine acellular dermal matrix (PADM) with or without an enamel matrix derivative (EMD) on gingival recession defects treated with a coronally advanced flap (CAF) in dogs. MATERIALS AND METHODS: Miller class II gingival recession defects (5 mm wide and 7 mm deep) were surgically created on the labial side of bilateral maxillary canines in 12 dogs. After 8 weeks of plaque accumulation, the 24 chronic defects were randomly assigned to one of the following 4 treatments: CAF, CAF with PADM (CAF/PADM), CAF with EMD (CAF/EMD), and CAF with EMD and PADM (CAF/EMD/PADM). The animals were sacrificed 10 weeks after surgery for histologic evaluation. RESULTS: In all groups, root coverage was obtained to a varying degree. PADM was well incorporated in gingival connective tissue in the CAF/PADM and in the CAF/EMD/PADM groups. The height of newly formed bone was significantly greater in the CAF/EMD/PADM group than in the CAF and CAF/PADM groups. New cementum with periodontal ligament-like tissue was predominantly found in the CAF/EMD and CAF/EMD/PADM groups. The CAF/EMD/PADM group showed the greatest amount of new cementum among the groups examined, although the difference was not statistically significant. CONCLUSION: Within the limitations of the present study, it can be concluded that CAF/EMD/PADM treatment may promote periodontal regeneration in gingival recession defects. CLINICAL RELEVANCE: The present results suggest that the combination of EMD and PADM in conjunction with CAF may represent a promising approach for treating single Miller class II gingival recessions.