Application of Hydroxycholesterols for Alveolar Cleft Osteoplasty in a Rodent Model.

BACKGROUND: Bone morphogenetic proteins (BMPs) have played a central role in the regenerative therapies for bone reconstruction, including alveolar cleft and craniofacial surgery. However, the high cost and significant adverse effect of BMPs limit their broad application. Hydroxycholesterols, naturally occurring products of cholesterol oxidation, are a promising alternative to BMPs. The authors studied the osteogenic capability of hydroxycholesterols on human mesenchymal stem cells and the impact of hydroxycholesterols on a rodent alveolar cleft model. METHODS: Human mesenchymal stem cells were treated with control medium or osteogenic medium with or without hydroxycholesterols. Evaluation of cellular osteogenic activity was performed. A critical-size alveolar cleft was created and one of the following treatment options was assigned randomly to each defect: collagen sponge incorporated with hydroxycholesterols, BMP-2, or no treatment. Bone regeneration was assessed by means of radiologic and histologic analyses and local inflammation in the cleft evaluated. Moreover, the role of the hedgehog signaling pathway in hydroxycholesterol-mediated osteogenesis was examined. RESULTS: All cellular osteogenic activities were significantly increased on human mesenchymal stem cells treated with hydroxycholesterols relative to others. The alveolar cleft treated with collagen sponge with hydroxycholesterols and BMP-2 demonstrated robust bone regeneration. The hydroxycholesterol group revealed histologically complete bridging of the alveolar defect with architecturally mature new bone. The inflammatory responses were less in the hydroxycholesterol group compared with the BMP-2 group. Induction of hydroxycholesterol-mediated in vitro osteogenesis and in vivo bone regeneration were attenuated by hedgehog signaling inhibitor, implicating involvement of the hedgehog signaling pathway. CONCLUSION: Hydroxycholesterols may represent a viable alternative to BMP-2 in bone tissue engineering for alveolar cleft.

Nitrogen-containing bisphosphonate therapy-Part II: Assessment of alveolar bone tissue inflammatory response in rats-A blind randomized controlled trial.

This study aimed to evaluate the alveolar bone tissue inflammatory response in rats undergoing zoledronic acid therapy. The study sample was composed of 28 Wistar rats. Animals from the test group GTa received a weekly intraperitoneal dose of 0.2 mg/kg of zoledronic acid for 3 weeks, while test group GTb received the same dose for 8 weeks. A physiological saline dose, equivalent to that of the medication, was administered to the controls in groups GCa and GCb. A defect was created in the dental crown of the lower first molars using a drill to simulate pulp and periapical injury. Data were evaluated regarding image grey levels by cone-beam computed tomography and histologically by assigning scores for the presence of inflammatory infiltrate, type of infiltrate, vascularization, bone necrosis and dental resorption. Grey levels in the 3-week therapy group (GTa) showed more pronounced changes in comparison with those seen in the GCa group (P < 0.05). Evaluation of the scores demonstrated no association between any of the variables amongst the groups (>0.05). However, bone remodelling decreased in the groups receiving the medication. Bone necrosis was present more frequently in group GTb than in the control group GCb. The results suggest that the drug interfered in the reaction capacity of the alveolar bone tissue as test group GTa showed higher grey levels in comparison to the control group GCa. In addition, there was less bone remodelling activity, with the appearance of bone necrosis zones and intense acute inflammatory infiltrate associated with the 8-week therapy group GTb.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Nitrogen-containing bisphosphonate therapy: assessment of the alveolar bone structure in rats - a blind randomized controlled trial.

This study aimed to assess the effect of zoledronic acid exposure on structures of the alveolar bone of rats. The sample was composed of 42 male Wistar rats. Animals in the T1 and T2 groups received weekly doses of 0.2 mg/kg intraperitoneal zoledronic acid for 3 weeks, while animals in the T3 group received the same treatment for 8 weeks. The control groups C1, C2 and C3 received equivalent doses of saline. The first upper molars of Wistar rats in the C2, T2, C3 and T3 groups were extracted. Cone-beam computerized tomography scans were performed, and the image density was analysed by grey levels. The presence and type of inflammatory infiltrate, vascularization and bone necrosis were assigned by histological qualitative scores. Histomorphometric analysis of bone density was performed in the groups without extraction. No significant differences were found in the bone grey density estimated by grey-level value and histomorphometric analysis between the C1 and T1 groups (P > 0.05). The grey levels in the T3 group were lower (P < 0.05) than in the C3 group, corresponding to the bone defect. Histological assessments showed the presence of bone necrosis in the T3 group and lower levels of bone remodelling in the test groups (T2 and T3) compared to the control groups (C2 and C3). The results of qualitative analyses did not differ significantly between the groups (P > 0.05). Zoledronic acid-exposed animals showed maxillary changes including reduced grey levels, the presence of bone necrosis and a higher prevalence of inflammatory signs.

Assessment of vascular endothelial growth factor and matrix metalloproteinase-9 in the periodontium of rats treated with atorvastatin.

BACKGROUND: The aim of this study is to examine, for the first time, the role of systemic and local atorvastatin application on periodontium using histomorphometric and immunohistochemical analysis during and after experimental periodontitis induction with or without the presence of microbial dental biofilm. METHODS: One hundred ten male Wistar rats were used. Silk ligatures were placed around the cervical area of the mandibular first molars; rats in the healthy control group received no ligatures (n = 10). In experimental periodontitis groups (n = 90), systemic and local atorvastatin and saline were administered in three different periods; the control periodontitis group (n = 10) received no treatment. Histomorphometric analysis, which included alveolar bone area, alveolar bone level, and attachment loss, and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9, were performed after the rats were sacrificed at the end of the experimental procedure. RESULTS: There was a greater increase in alveolar bone area and VEGF immunoreactivity, as well as a greater decrease in alveolar bone and attachment loss and MMP-9 immunoreactivity, with systemic and local atorvastatin application during and after induction of experimental periodontitis. Local atorvastatin application showed better results on periodontium with regard to alveolar bone findings. CONCLUSIONS: Systemic and local atorvastatin application showed beneficial effects on periodontium during and after induction of experimental periodontitis. Within the limits of this study, it can be concluded that atorvastatin, which is used for hypercholesterolemia treatment, can also be used as a protective and therapeutic agent for periodontal disease.

Assessment of hemostatic efficacy and osseous wound healing using HemCon dental dressing.

INTRODUCTION: Obtaining hemostasis in the surgical crypt during periradicular surgery is essential. It allows for improved visibility and contributes to a dry environment suitable for the placement of moisture-sensitive root-end filling material. Although current materials may not be moisture sensitive during setting, hemostasis is important for proper placement of root-end filling materials during apical surgery. A new hemostatic agent, HemCon dental dressing (Patterson Dental, St Paul, MN), may improve upon the efficacy of wound healing and hemostasis both in extent and time. The aim of this study was to evaluate the hemostatic effect of HemCon in osseous wound sites and evaluate the wound healing potential and percentage of new bone formation in osseous crypts treated with HemCon. METHODS: A split-mouth design was used with random allocation of sham and experimental sites in 12 rabbits. In experimental sites, either HemCon or 15.5% ferric sulfate was applied to osseous crypts created with a round bur. Hemostatic efficacy was evaluated using predetermined scores. Rabbits were sacrificed at 21 days, and tissues were harvested and prepared for histologic evaluation. A blinded pathologist scored samples relative to inflammation. The percentage of new bone deposition was calculated using NIS Elements software (Nikon Instruments Inc, Melville, NY). RESULTS: There was no statistical significant difference in hemostatic efficacy or wound healing between HemCon and ferric sulfate (P > .05). The HemCon group showed a significantly higher percentage of new bone deposition compared with the controls (P < .01). CONCLUSIONS: HemCon shows promise as an adjunct to the endodontic surgical armamentarium.

Assessment of the potential of growth factors for localized alveolar ridge augmentation: a systematic review.

OBJECTIVE: To systematically assess the literature regarding the clinical, histological, and radiographic outcome of bone morphogenetic proteins (BMP-2, BMP-7), growth/differentiation factor-5 (GDF-5), platelet-derived growth factor (PDGF), and parathyroid hormone (PTH) for localized alveolar ridge augmentation. MATERIAL AND METHODS: Five separate Medline searches were performed in duplicate for human and animal studies, respectively. The primary outcome of the included studies was bone regeneration of localized alveolar ridge defects or craniofacial defects. RESULTS: In six human studies, BMP-2 affected local bone augmentation with increasing volume for higher doses. A majority (43 of 45) of animal studies using BMP-2 showed a positive effect in favour of the growth factor (GF). In six of eight studies, a positive effect was associated with the use of BMP-7. Only one animal study was included for GDF-5 revealing statistically significantly higher bone volume. Regarding PDGF, statistically significantly higher bone volume was observed in five of 10 included studies. Four animal studies using PTH revealed statistically significantly more bone regeneration compared with controls. CONCLUSIONS: Differing levels and quantity of evidence were noted to be available for the GFs evaluated, revealing that BMP-2, BMP-7, GDF-5, PDGF, and PTH may stimulate local bone augmentation to various degrees. Human data for the potential of rhBMP-2 are supportive.