Genome-wide identification of walnut (Juglans regia) PME gene family members and expression analysis during infection with Cryptosphaeria pullmanensis pathogens.

Walnuts exhibit a higher resistance to diseases, though they are not completely immune. This study focuses on the Pectin methylesterase (PME) gene family to investigate whether it is involved in disease resistance in walnuts. These 21 genes are distributed across 12 chromosomes, with four pairs demonstrating homology. Variations in conserved motifs and gene structures suggest diverse functions within the gene family. Phylogenetic and collinear gene pairs of the PME family indicate that the gene family has evolved in a relatively stable way. The cis-acting elements and gene ontology enrichment of these genes, underscores their potential role in bolstering walnuts' defense mechanisms. Transcriptomic analyses were conducted under conditions of Cryptosphaeria pullmanensis infestation and verified by RT-qPCR. The results showed that certain JrPME family genes were activated in response, leading to the hypothesis that some members may confer resistance to the disease.

Synergistic effect of biomass and polyurethane waste co-pyrolysis on soot formation at high temperatures.

Soot is an important toxic pollutant generated during high-temperature incineration of solid waste (i.e., biomass and plastic waste) under air-lean conditions, and has a great impact on flame radiation. The main objective of this work is to study the synergistic effect of biomass and polyurethane co-pyrolysis on soot formation at high temperatures (1100-1250 °C). The effects of temperature, biomass species, and co-pyrolysis ratio on the yield, morphology, composition and reactivity of soot particles are studied. Results show that under controlled co-pyrolysis conditions, the measured soot yield from co-pyrolysis of biomass and polyurethane is lower than the theoretical value by weight average, while the particle size distribution tends to concentrate on a smaller diameter range. The degree of synergistic effect increases with the increasing biomass ratio (0-50 wt%) and decreasing pyrolysis temperature. Wood in co-pyrolysis presents a stronger synergistic effect on soot yields than straw co-pyrolysis does. Degree of synergistic effect on soot oxidation reactivity depends much on the biomass addition ratio but less on pyrolysis temperature. At 10 wt% straw addition ratio, co-pyrolysis exerts a negative synergistic effect on soot oxidation reactivity, while the synergistic effect turns significantly positive when the straw addition ratio increases to 50 wt%.

Platelet-Derived Growth Factor Delivery via Nanofibrous Scaffolds for Soft-Tissue Repair.

BACKGROUND: Platelet-derived growth factor (PDGF) is a multifunctional growth factor that exerts its biological effects on cellular chemotaxis, proliferation, matrix synthesis, antiapoptosis, and vascularization. PDGF is clinically approved to treat neuropathic diabetic ulcers and osseous defects due to periodontal disease. THE PROBLEM: The short half-life in vivo of PDGF limits the efficacy of its biological functions. Solving this problem remains a key obstacle for PDGF clinical application. Therefore, the development of an optimized controlled release delivery system offers significant potential. BASIC/CLINICAL SCIENCE ADVANCES: In this article, we highlight the development of a polymeric delivery system of nanofibrous scaffolds containing PDGF-encapsulated microspheres for tissue engineering. The designed scaffolds were evaluated in a subcutaneous implantation model for tissue neogenesis, vascularization, and chemokine gene expression, as well as soft-tissue repair. PDGF was found to strongly upregulate in vivo gene expression of the CXC chemokine family members such as CXC chemokine ligand CXCL1, CXCL2, and CXCL5 that are important in angiogenesis, inflammation, and wound repair. CLINICAL CARE RELEVANCE: Recombinant human PDGF is approved by the Food and Drug Administration for patients afflicted with diabetic foot ulcers or compromised periodontal wounds. Challenges related to the transient biological activity of bolus PDGF administration using currently available release systems continue. Thus, it is necessary to explore new delivery systems to optimize biological activity and bioavailability of tissue growth factors. CONCLUSION: The use of a controlled, "dial-able" delivery system allows for a more tightly regulated release of factors to promote repair of soft- and hard-tissue defects for clinical application.

Bone loss in the anterior edentulous maxilla opposing two-implant-supported overdentures vs complete dentures: a systematic review and meta-analysis.

OBJECTIVE: To investigate bone loss in the anterior edentulous maxilla restored with maxillary complete dentures and opposed by mandibular two-implant-supported overdentures (2-IODs) or complete dentures. METHOD AND MATERIALS: A systematic search was conducted using the Ovid MEDLINE, Embase, Web of Science, CINAHL, and Cochrane databases for studies investigating bone loss in the anterior edentulous maxilla with mandibular 2-IODs or complete dentures. Two reviewers assessed the eligibility of studies and risk of bias assessment was conducted according to the Newcastle-Ottawa Scale. A meta-analysis was performed using statistical software to estimate weighted mean difference in bone loss with 95% confidence interval (CI). The level of significance was defined as P value (< .05). RESULTS: A total of 2,510 studies were identified through electronic and manual searching. Six studies were selected and compounded for quantitative synthesis of 163 patients. Bone loss in the anterior edentulous maxilla was greater with 2-IODs than with complete dentures. The total estimate of weighted mean difference between 2-IODs and complete dentures was -1.40 (95% CI -3.12 to 0.31). However, the difference was not statistically significant (P = .11). The data were heterogenous across the studies based on chi-square statistics (χ2 [df = 7] = 52.75, P < .0001; τ2 = 5.53, I2 = 95.21%). In addition, the impact of implant splinting on bone loss was not significant (P > .29). None of the included studies were considered to be at high risk of bias. CONCLUSION: Within the limitations of the current systematic review and meta-analysis, the estimate of bone loss in the anterior edentulous maxilla was greater with 2-IODs than with complete dentures. However, the difference was not statistically significant. A well-designed randomized clinical study needs to be conducted to validate the results of this systematic review.

Characterization of ligature-induced experimental periodontitis.

We sought to better characterize the progression of periodontal tissue breakdown in rats induced by a ligature model of experimental periodontal disease (PD). A total of 60 male Sprague-Dawley rats were evenly divided into an untreated control group and a PD group induced by ligature bilaterally around first and second maxillary molars. Animals were sacrificed at 1, 3, 5, 7, 14, and 21 days after the induction of PD. Alveolar bone loss was evaluated by histomorphometry and microcomputed tomography (µCT). The immune-inflammatory process in the periodontal tissue was assessed using descriptive histologic analysis and quantitative polymerase chain reaction (qPCR). This ligature model resulted in significant alveolar bone loss and increased inflammatory process of the periodontal tissues during the initial periods of evaluation (0-14 days). A significant increase in the gene expression of pro-inflammatory cytokines, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and proteins involved in osteoclastogenesis, receptor activator of nuclear factor-k B ligand (RANKL) and osteoprotegerin (OPG) was observed in the first week of analysis. In the later periods of evaluation (14-21 days), no significant alterations were noted with regard to inflammatory processes, bone resorption, and expression of cytokine genes. The ligature-induced PD model resulted in progressive alveolar bone resorption with two different phases: Acute (0-14 days), characterized by inflammation and rapid bone resorption, and chronic (14-21 days) with no significant progression of bone loss. Furthermore, the gene expressions of IL-6, IL-1ß, TNF-α, RANKL, and OPG were highly increased during the progress of PD in the early periods. RESEARCH HIGHLIGHTS: Ligature-induced bone resorption in rats occurred in the initial periods after disease induction The bone resorption was characterized by two distinct phases: Acute (0-14 days), with pronounced inflammation and alveolar bone loss Chronic phase (14-21 days): No further disease progression Several pro-inflammatory cytokines were increased during the progress of periodontitis.

Sclerostin-Neutralizing Antibody Enhances Bone Regeneration Around Oral Implants.

BACKGROUND: Dental implants are an important option for replacement of missing teeth. A major clinical challenge is how best to accelerate bone regeneration and reduce the healing time for functional restoration after implant placement. A sclerostin-neutralizing antibody (Scl-Ab) has been shown to enhance alveolar bone formation and fracture repair. The aim of this study was to investigate the effects of systemic administration of Scl-Ab on dental implant osseointegration and bone regeneration in an experimental alveolar ridge tooth extraction model. MATERIALS AND METHODS: To investigate the effects of Scl-Ab on bone regeneration and dental implant osseointegration, an experimental alveolar bone osteotomy rat model was adopted. One month after extraction of maxillary right first molars, osteotomy defects were created at the coronal aspect of each of the extraction sites, and 1 × 2-mm custom titanium implants were installed into the osteotomies. Coincident with implant placement, Scl-Ab was administered subcutaneously at a dose of 25 mg/kg twice weekly for 10-28 days and compared with a vehicle control. Animals were sacrificed 10, 14, and 28 days after surgery, and maxillae were harvested and analyzed by microcomputed tomography (microCT), histology, and histomorphometry. RESULTS: microCT analysis demonstrated that the maxillary bone volume fraction was approximately 2- to 2.5-fold greater in Scl-Ab-treated animals compared with vehicle alone at days 14 and 28. Consistent with those findings, two-dimensional bone fill percentages within the coronal osteotomy sites were highest in Scl-Ab treatment groups at 28 days. In addition, bone-implant contact at 28 days was approximately twofold greater in the Scl-Ab group compared with the vehicle control. CONCLUSIONS: These results indicate that systemic Scl-Ab administration enhances osseointegration and bone regeneration around dental implants. This approach offers potential as a treatment modality for patients with low bone mass or bone defects to achieve more predictable bone regeneration at alveolar bone defects and to enhance dental implant osseointegration.

The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres.

It is advantageous to incorporate controlled growth factor delivery into tissue engineering strategies. The objective of this study was to develop a three-dimensional (3D) porous tissue engineering scaffold with the capability of controlled releasing recombinant human bone morphogenetic protein-7 (rhBMP-7) for enhancement of bone regeneration. RhBMP-7 was first encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanospheres (NS) with an average diameter of 300nm. Poly(l-lactic acid) (PLLA) scaffolds with interconnected macroporous and nano-fibrous architectures were prepared using a combined sugar sphere template leaching and phase separation technique. A post-seeding technique was then utilized to immobilize rhBMP-7 containing PLGA nanospheres onto prefabricated nano-fibrous PLLA scaffolds with well-maintained 3D structures. In vitro release kinetics indicated that nanosphere immobilized scaffold (NS-scaffold) could release rhBMP-7 in a temporally controlled manner, depending on the chemical and degradation properties of the NS which were immobilized onto the scaffold. In vivo, rhBMP-7 delivered from NS-scaffolds induced significant ectopic bone formation throughout the scaffold while passive adsorption of rhBMP-7 into the scaffold resulted in failure of bone induction due to either the loss of rhBMP-7 biological function or insufficient duration within the scaffold. We conclude that the interconnected macroporous architecture and the sustained, prolonged delivery of bioactive rhBMP-7 from NS immobilized nano-fibrous scaffolds actively induced new bone formation throughout the scaffold. The approach offers a new delivery method of BMPs and a novel scaffold design for bone regeneration.

Three-dimensional micro-computed tomographic imaging of alveolar bone in experimental bone loss or repair.

BACKGROUND: Micro-computed tomography (micro-CT) offers significant potential for identifying mineralized structures. However, three-dimensional (3-D) micro-CT of alveolar bone has not been adapted readily for quantification. Moreover, conventional methods are not highly sensitive for analyzing bone loss or bone gain following periodontal disease or reconstructive therapy. The objective of this investigation was to develop a micro-CT methodology for quantifying tooth-supporting alveolar bone in 3-D following experimental preclinical situations of periodontitis or reconstructive therapy. METHODS: Experimental in vivo bone loss or regeneration situations were developed to validate the micro-CT imaging techniques. Twenty mature Sprague-Dawley rats were divided into two groups: bone loss (Porphyromonas gingivalis lipopolysaccharide-mediated bone resorption) and regenerative therapy. Micro-CT and software digitized specimens were reconstructed three-dimensionally for linear and volumetric parameter assessment of alveolar bone (linear bone height, bone volume, bone volume fraction, bone mineral content, and bone mineral density). Intra- and interexaminer reproducibility and reliability were compared for methodology validation. RESULTS: The results demonstrated high examiner reproducibility for linear and volumetric parameters with high intraclass correlation coefficient (ICC) and coefficient of variation (CV). The ICC showed that the methodology was highly reliable and reproducible (ICC >0.99; 95% confidence interval, 0.937 to 1.000; CV <1.5%), suggesting that 3-D measurements may provide better alveolar bone analysis than conventional 2-D methods. CONCLUSIONS: The developed methods allow for highly accurate and reproducible static measurements of tooth-supporting alveolar bone following preclinical situations of bone destruction or regeneration. Future investigations should focus on using in vivo micro-CT imaging for real-time assessments of alveolar bone changes.

RANKL inhibition through osteoprotegerin blocks bone loss in experimental periodontitis.

BACKGROUND: Prevention of alveolar bone destruction is a clinical challenge in periodontal disease treatment. The receptor activator of nuclear factor-kappa B ligand (RANKL) inhibitor osteoprotegerin (OPG) inhibits osteoclastogenesis and suppresses bone resorption. METHODS: To study the effects of RANKL inhibition on alveolar bone loss, an experimental ligature-induced model of periodontitis was used. A total of 32 rats were administered human OPG-Fc fusion protein (10 mg/kg) or vehicle by subcutaneous delivery twice weekly for 6 weeks. Negative or positive controls received no treatment or disease through vehicle delivery, respectively. Biopsies were harvested after 3 and 6 weeks, and mandibulae were evaluated by microcomputed tomography (microCT) and histology. Serum levels of human OPG-Fc and tartrate-resistant acid phosphatase-5b (TRAP-5b) were measured throughout the study by enzyme-linked immunosorbent assay (ELISA). Statistical analyses included analysis of variance (ANOVA) and Tukey tests. RESULTS: Human OPG-Fc was detected in the sera of OPG-Fc-treated animals by 3 days and throughout the study. Serum TRAP-5b was sharply decreased by OPG-Fc treatment soon after OPG-Fc delivery and remained low for the observation period. Significant preservation of alveolar bone volume was observed among OPG-Fc-treated animals compared to the controls at weeks 3 and 6 (P <0.05). Descriptive histology revealed that OPG-Fc significantly suppressed osteoclast surface area at the alveolar crest. CONCLUSION: Systemic delivery of OPG-Fc inhibits alveolar bone resorption in experimental periodontitis, suggesting that RANKL inhibition may represent an important therapeutic strategy for the prevention of progressive alveolar bone loss.

Chemotherapy-Induced Depletion of OCT4-Positive Cancer Stem Cells in a Mouse Model of Malignant Testicular Cancer.

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy.

Effect of rhPDGF-BB delivery on mediators of periodontal wound repair.

Growth factors such as platelet-derived growth factor (PDGF) exert potent effects on wound healing including the regeneration of tooth-supporting structures. This investigation examined the effect of the local delivery of PDGF-BB when combined with reconstructive periodontal surgery on local wound fluid (WF) levels of PDGF-AB, vascular endothelial growth factor (VEGF), and bone collagen telopeptide (ICTP) in humans with advanced periodontitis. Sixteen patients exhibiting localized periodontal osseous defects were randomized to one of three groups (beta-TCP carrier alone, beta-TCP + 0.3 mg/mL of recombinant human PDGF-BB [rhPDGF-BB], or beta-TCP + 1.0 mg/mL of rhPDGF-BB) and monitored for 6 months. WF was harvested and analyzed for PDGF-AB, VEGF, and ICTP WF levels. Teeth contralateral to the target lesions served as controls. Increased levels of VEGF in the WF was observed for all surgical treatment groups with the 1.0 mg/mL rhPDGF-BB group showing the most pronounced difference at 3 weeks in the AUC analysis versus control (p < 0.0001). PDGF-AB WF levels were increased for the carrier alone group compared to both rhPDGFBB groups. Low-dose rhPDGF-BB application elicited increases in ICTP at days 3-5 in the wound healing process, suggesting a promotion of bone turnover at early stages of the repair process (p < 0.02). These results demonstrate contrasting inducible expression patterns of PDGF-AB, VEGF, and ICTP during periodontal wound healing in humans.

Nano-fibrous scaffold for controlled delivery of recombinant human PDGF-BB.

The localized and temporally controlled delivery of growth factors is key to achieving optimal clinical efficacy. In sophisticated tissue engineering strategies, the biodegradable scaffold is preferred to serve as both a three-dimensional (3-D) substrate and a growth factor delivery vehicle to promote cellular activity and enhance tissue neogenesis. This study presents a novel approach to fabricate tissue engineering scaffolds capable of controlled growth factor delivery whereby growth factor containing microspheres were incorporated into 3-D scaffolds with good mechanical properties, well-interconnected macroporous and nano-fibrous structures. The microspheres were uniformly distributed throughout the nano-fibrous scaffold and their incorporation did not interfere the macro-, micro-, and nanostructures of the scaffold. The release kinetics of platelet-derived growth factor-BB (PDGF-BB) from microspheres and scaffolds was investigated using poly(lactic-co-glycolic acid) (PLGA50) microspheres with different molecular weights (6.5 and 64kDa, respectively) and microsphere-incorporated poly(l-lactic acid) (PLLA) nano-fibrous scaffolds. Incorporation of microspheres into scaffolds significantly reduced the initial burst release. Sustained release from several days to months was achieved through different microspheres in scaffolds. Released PDGF-BB was demonstrated to possess biological activity as evidenced by stimulation of human gingival fibroblast DNA synthesis in vitro. The successful generation of 3-D nano-fibrous scaffold incorporating controlled-release factors indicates significant potential for more complex tissue regeneration.

Gene therapeutics for periodontal regenerative medicine.

There has been significant advancement in the field of periodontal tissue engineering over the past decade for the repair of tooth-supporting structures. Although encouraging results for periodontal tissue regeneration have been found in numerous clinical investigations using recombinant growth factors, limitations exist with topical protein delivery. Newer approaches seek to develop methodologies that optimize growth factor targeting to maximize the therapeutic outcome of periodontal regenerative procedures. Genetic approaches in periodontal tissue engineering show early progress in achieving delivery of growth factor genes, such as platelet-derived growth factor or bone morphogenetic protein, to periodontal lesions. Ongoing investigations in ex vivo and in vivo gene transfer to periodontia seek to examine the extent of the potential effects in stimulating periodontal tissue engineering.

Platelet-derived growth factor gene delivery stimulates ex vivo gingival repair.

Destruction of tooth support due to the chronic inflammatory disease periodontitis is a major cause of tooth loss. There are limitations with available treatment options to tissue engineer soft tissue periodontal defects. The exogenous application of growth factors (GFs) such as platelet-derived growth factor (PDGF) has shown promise to enhance oral and periodontal tissue regeneration. However, the topical administration of GFs has not led to clinically significant improvements in tissue regeneration because of problems in maintaining therapeutic protein levels at the defect site. The utilization of PDGF gene transfer may circumvent many of the limitations with protein delivery to soft tissue wounds. The objective of this study was to test the effect of PDGF-A and PDGF-B gene transfer to human gingival fibroblasts (HGFs) on ex vivo repair in three-dimensional collagen lattices. HGFs were transduced with adenovirus encoding PDGF-A and PDGF-B genes. Defect fill of bilayer collagen gels was measured by image analysis of cell repopulation into the gingival defects. The modulation of gene expression at the defect site and periphery was measured by RT-PCR during a 10-day time course after gene delivery. The results demonstrated that PDGF-B gene transfer stimulated potent (>4-fold) increases in cell repopulation and defect fill above that of PDGF-A and corresponding controls. PDGF-A and PDGF-B gene expression was maintained for at least 10 days. PDGF gene transfer upregulated the expression of phosphatidylinosital 3-kinase and integrin alpha5 subunit at 5 days after adenovirus transduction. These results suggest that PDGF gene transfer has potential for periodontal soft tissue-engineering applications.

Cementoblast delivery for periodontal tissue engineering.

BACKGROUND: Predictable periodontal regeneration following periodontal disease is a major goal of therapy. The objective of this proof of concept investigation was to evaluate the ability of cementoblasts and dental follicle cells to promote periodontal regeneration in a rodent periodontal fenestration model. METHODS: The buccal aspect of the distal root of the first mandibular molar was denuded of its periodontal ligament (PDL), cementum, and superficial dentin through a bony window created bilaterally in 12 athymic rats. Treated defects were divided into three groups: 1) carrier alone (PLGA polymer sponges), 2) carrier + follicle cells, and 3) carrier + cementoblasts. Cultured murine primary follicle cells and immortalized cementoblasts were delivered to the defects via biodegradable PLGA polymer sponges, and mandibulae were retrieved 3 weeks and 6 weeks post-surgery for histological evaluation. In situ hybridization, for gene expression of bone sialoprotein (BSP) and osteocalcin (OCN), and histomorphometric analysis were further done on 3-week specimens. RESULTS: Three weeks after surgery, histology of defects treated with carrier alone indicated PLGA particles, fibrous tissue, and newly formed bone scattered within the defect area. Defects treated with carrier + follicle cells had a similar appearance, but with less formation of bone. In contrast, in defects treated with carrier + cementoblasts, mineralized tissues were noted at the healing site with extension toward the root surface, PDL region, and laterally beyond the buccal plate envelope of bone. No PDL-bone fibrous attachment was observed in any of the groups at this point. In situ hybridization showed that the mineralized tissue formed by cementoblasts gave strong signals for both BSP and OCN genes, confirming its nature as cementum or bone. The changes noted at 3 weeks were also observed at 6 weeks. Cementoblast-treated and carrier alone-treated defects exhibited complete bone bridging and PDL formation, whereas follicle cell-treated defects showed minimal evidence of osteogenesis. No new cementum was formed along the root surface in the above two groups. Cementoblast-treated defects were filled with trabeculated mineralized tissue similar to, but more mature, than that seen at 3 weeks. Furthermore, the PDL region was maintained with well-organized collagen fibers connecting the adjacent bone to a thin layer of cementum-like tissue observed on the root surface. Neoplastic changes were observed at the superficial portions of the implants in two of the 6-week cementoblast-treated specimens, possibly due in part to the SV40-transformed nature of the implanted cell line. CONCLUSIONS: This pilot study demonstrates that cementoblasts have a marked ability to induce mineralization in periodontal wounds when delivered via polymer sponges, while implanted dental follicle cells seem to inhibit periodontal healing. These results confirm the selective behaviors of different cell types in vivo and support the role of cementoblasts as a tool to better understand periodontal regeneration and cementogen-

Effect of sustained gene delivery of platelet-derived growth factor or its antagonist (PDGF-1308) on tissue-engineered cementum.

BACKGROUND: Cementum, a mineralized tissue lining the tooth root surface, is destroyed during the inflammatory process of periodontitis. Restoration of functional cementum is considered a criterion for successful regeneration of periodontal tissues, including formation of periodontal ligament, cementum, and alveolar bone. Short-term administration of platelet-derived growth factor (PDGF) has been shown to partially regenerate periodontal structures. Nonetheless, the role of PDGF in cementogenesis is not well understood. The aim of the present study was to determine the effect of sustained PDGF gene transfer on cementum formation in an ex vivo ectopic biomineralization model. METHODS: Osteocalcin (OC) promoter-driven SV40 transgenic mice were used to obtain immortalized cementoblasts (OCCM). The OCCM cells were transduced with adenoviruses (Ad) encoding either PDGF-A, an antagonist of PDGF signaling (PDGF-1308), a control virus (green fluorescent protein, GFP), or no treatment (NT). The transduced cells were incorporated into polymer scaffolds and implanted subcutaneously into severe combined immunodeficient (SCID) mice. The implants were harvested at 3 and 6 weeks for histomorphometric analysis of the newly formed mineralized tissues. Northern blot analysis was performed to determine the expression levels of mineral-associated genes including bone sialoprotein (BSP), OC, and osteopontin (OPN) in the cell-implant specimens at 3 and 6 weeks. RESULTS: The results indicated mineralization was significantly reduced in both the Ad/PDGF-A and Ad/PDGF-1308 treated specimens when compared to the NT or Ad/GFP groups at 3 and 6 weeks (P<0.01). In addition, the size of the implants treated with Ad/PDGF-A and Ad/PDGF-1308 was significantly reduced compared to implants from Ad/GFP and NT groups at 3 weeks (P<0.05). At 6 weeks, the size of implants and mineral formation increased in NT, Ad/GFP, and Ad/PDGF-A groups, while the Ad/PDGF-1308 treated implants continued to decrease in size and mineral formation (P<0.01). Northern blot analysis revealed that in the Ad/PDGF-A treated implants OPN was increased, whereas OC gene expression was downregulated at 3 weeks. In the Ad/PDGF-1308 treated implants, BSP, OC, and OPN were all downregulated at 3 weeks. At 3 weeks, the Ad/PDGF-A treated implants contained significantly higher multinucleated giant cell (MNGC) density compared to NT, Ad/GFP, and Ad/PDGF-1308 specimens. The MNGC density in NT, Ad/GFP, and Ad/PDGF-A treated groups reduced over time, while the Ad/PDGF-1308 transduced implants continued to exhibit significantly higher MNGC density compared with the other treatment groups at 6 weeks. CONCLUSIONS: The results showed that continuous exposure to PDGF-A had an inhibitory effect on cementogenesis, possibly via the upregulation of OPN and subsequent enhancement of MNGCs at 3 weeks. On the other hand, Ad/PDGF-1308 inhibited mineralization of tissue-engineered cementum possibly due to the observed downregulation of BSP and OC and a persistence of stimulation of MNGCs. These findings suggest that continuous exogenous delivery of PDGF-A may delay mineral formation induced by cementoblasts, while PDGF is clearly required for mineral neogenesis.

SDF-1 enhances wound healing of critical-sized calvarial defects beyond self-repair capacity.

Host blood circulating stem cells are an important cell source that participates in the repair of damaged tissues. The clinical challenge is how to improve the recruitment of circulating stem cells into the local wound area and enhance tissue regeneration. Stromal-derived factor-1 (SDF-1) has been shown to be a potent chemoattractant of blood circulating stem cells into the local wound microenvironment. In order to investigate effects of SDF-1 on bone development and the repair of a large bone defect beyond host self-repair capacity, the BMP-induced subcutaneous ectopic bone formation and calvarial critical-sized defect murine models were used in this preclinical study. A dose escalation of SDF-1 were loaded into collagen scaffolds containing BMP, VEGF, or PDGF, and implanted into subcutaneous sites at mouse dorsa or calvarial critical-sized bone defects for 2 and 4 weeks. The harvested biopsies were examined by microCT and histology. The results demonstrated that while SDF-1 had no effect in the ectopic bone model in promoting de novo osteogenesis, however, in the orthotopic bone model of the critical-sized defects, SDF-1 enhanced calvarial critical-sized bone defect healing similar to VEGF, and PDGF. These results suggest that SDF-1 plays a role in the repair of large critical-sized defect where more cells are needed while not impacting de novo bone formation, which may be associated with the functions of SDF-1 on circulating stem cell recruitment and angiogenesis.

Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

Sclerostin antibody stimulates bone regeneration after experimental periodontitis.

The reconstruction of large osseous defects due to periodontitis is a challenge in regenerative therapy. Sclerostin, secreted by osteocytes, is a key physiological inhibitor of osteogenesis. Pharmacologic inhibition of sclerostin using sclerostin-neutralizing monoclonal antibody (Scl-Ab) thus increases bone formation, bone mass and bone strength in models of osteopenia and fracture repair. This study assessed the therapeutic potential of Scl-Ab to stimulate alveolar bone regeneration following experimental periodontitis (EP). Ligature-induced EP was induced in rats to generate localized alveolar bone defects. Following 4 weeks of disease induction, Scl-Ab (+EP) or vehicle (+/- EP) were systemically delivered, twice weekly for up to 6 wks to determine the ability of Scl-Ab to regenerate bone around tooth-supporting osseous defects. 3 and 6 wks after the initiation of Scl-Ab or vehicle treatment, femur and maxillary jawbones were harvested for histology, histomorphometry, and micro-computed tomography (micro-CT) of linear alveolar bone loss (ABL) and volumetric measures of bone support, including bone volume fraction (BVF) and tissue mineral density (TMD). Serum was analyzed to examine bone turnover markers during disease and regenerative therapy. Vehicle + EP animals exhibited maxillary bone loss (BVF, TMD and ABL) at ligature removal and thereafter. 6 weeks of Scl-Ab significantly improved maxillary bone healing, as measured by BVF, TMD and ABL, when compared to vehicle + EP. After 6 weeks of treatment, BVF and TMD values in the Scl-Ab + EP group were similar to those of healthy controls. Serum analysis demonstrated higher levels of bone formation markers osteocalcin and PINP in Scl-Ab treatment groups. Scl-Ab restored alveolar bone mass following experimental periodontitis. These findings warrant further exploration of Scl-Ab therapy in this and other oral bone defect disease scenarios.

Methods to validate tooth-supporting regenerative therapies.

In humans, microbially induced inflammatory periodontal diseases are the primary initiators that disrupt the functional and structural integrity of the periodontium (i.e., the alveolar bone, the periodontal ligament, and the cementum). The reestablishment of its original structure, properties, and function constitutes a significant challenge in the development of new therapies to regenerate tooth-supporting defects. Preclinical models represent an important in vivo tool to critically evaluate and analyze the key aspects of novel regenerative therapies, including (1) safety, (2) effectiveness, (3) practicality, and (4) functional and structural stability over time. Therefore, these models provide foundational data that supports the clinical validation and the development of novel innovative regenerative periodontal technologies. Steps are provided on the use of the root fenestration animal model for the proper evaluation of periodontal outcome measures using the following parameters: descriptive histology, histomorphometry, immunostaining techniques, three-dimensional imaging, electron microscopy, gene expression analyses, and safety assessments. These methods will prepare investigators and assist them in identifying the key end points that can then be adapted to later stage human clinical trials.