Periodontal Fenestration Defects Research Articles

Lacticaseibacillus rhamnosus GG Improves Periodontal Bone Repair via Gut–Blood Axis in Hyperlipidemia

Periodontal bone regeneration remains a clinical challenge, and hyperlipidemia can aggravate alveolar bone resorption. Probiotics have recently been reported to improve bone mass. We aimed to determine the role of Lacticaseibacillus rhamnosus GG (LGG) in periodontal bone regeneration improvement within the context of periodontitis with hyperlipidemia. A Sprague Dawley rat model for periodontitis, hyperlipidemia, and periodontal fenestration defect was constructed (n = 36) and administered LGG gavage for 6 wk (the rats were subsequently sacrificed). Fecal microbiota from donor rats 3 wk after LGG gavage was transplanted into recipient rats to evaluate the role of LGG-modulated gut microbiota in periodontal bone regeneration. Regenerated bone mass was detected using micro–computerized tomography and hematoxylin and eosin stain. Gut microbiota was analyzed using 16S ribosomal RNA sequencing. Serum metabolites were detected by liquid chromatography–mass spectrometry (6 wk after LGG gavage). The pro-osteogenic effects of screened serum metabolite were verified in vitro on bone marrow mesenchymal stem cells (BMMSCs). We found that the bone mineral density, bone volume (BV), trabecular bone volume fraction (BV/TV), and trabecular thickness of the regenerated periodontal bone increased after LGG gavage (P < 0.05) but had little effect on oral flora. After LGG gavage, Staphylococcus, Corynebacterium, and Collinsella in the gut of donors were significantly changed, and these differences were maintained in recipients, who also showed increased trabecular thickness of the regenerated periodontal bone (P < 0.05). These key genera were correlated with BV/TV and BV (P < 0.05). In addition, LGG gavage significantly regulated bone-related blood metabolites, of which selenomethionine promoted BMMSC osteogenesis. Notably, selenomethionine was associated with key gut genera (P < 0.05). Collectively, LGG improved periodontal bone regeneration in the context of periodontitis with hyperlipidemia by modulating gut microbiota and increasing pro-osteogenic metabolites in the blood. These results reveal new insights into the use of probiotics to promote periodontal bone regeneration via the gut–blood–bone axis.

Cementum protein 1 gene-modified adipose-derived mesenchymal stem cell sheets enhance periodontal regeneration in osteoporosis rat.

Osteoporosis (OP) and periodontitis are both diseases with excessive bone resorption, and the number of patients who suffer from these diseases is expected to increase. OP has been identified as a risk factor that accelerates the pathological process of periodontitis. Achieving effective and safe periodontal regeneration in OP patients is a meaningful challenge. This study aimed to assess the efficacy and biosecurity of human cementum protein 1 (hCEMP1) gene-modified cell sheets for periodontal fenestration defect regeneration in an OP rat model. Rat adipose-derived mesenchymal stem cells (rADSCs) were isolated from Sprague-Dawley rats. After primary culture, rADSCs were subjected to cell surface analysis and multi-differentiation assay. And rADSCs were transduced with hCEMP1 by lentiviral vector, and hCEMP1 gene-modified cell sheets were generated. The expression of hCEMP1 was evaluated by reverse transcription polymerase chain reaction and immunocytochemistry staining, and transduced cell proliferation was evaluated by Cell Counting Kit-8. The hCEMP1 gene-modified cell sheet structure was detected by histological analysis and scanning electron microscopy. Osteogenic and cementogenic-associated gene expression was evaluated by real-time quantitative polymerase chain reaction. In addition, an OP rat periodontal fenestration defect model was used to evaluate the regeneration effect of hCEMP1 gene-modified rADSC sheets. The efficacy was assessed with microcomputed tomography and histology, and the biosecurity of gene-modified cell sheets was evaluated by histological analysis of the spleen, liver, kidney and lung. The rADSCs showed a phenotype of mesenchymal stem cells and possessed multi-differentiation capacity. The gene and protein expression of hCEMP1 through lentiviral transduction was confirmed, and there was no significant effect on rADSC proliferation. Overexpression of hCEMP1 upregulated osteogenic and cementogenic-related genes such as runt-related transcription factor 2, bone morphogenetic protein 2, secreted phosphoprotein 1 and cementum attachment protein in the gene-modified cell sheets. The fenestration lesions in OP rats treated with hCEMP1 gene-modified cell sheets exhibited complete bone bridging, cementum and periodontal ligament formation. Furthermore, histological sections of the spleen, liver, kidney and lung showed no evident pathological damage. This pilot study demonstrates that hCEMP1 gene-modified rADSC sheets have a marked ability to enhance periodontal regeneration in OP rats. Thus, this approach may represent an effective and safe strategy for periodontal disease patients with OP.

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.

Sequential anti-inflammatory and osteogenic effects of a dual drug delivery scaffold loaded with parthenolide and naringin in periodontitis.

Our pilot study showed that a 3-dimensional dual drug delivery scaffold (DDDS) loaded with Chinese herbs significantly increased the regenerated bone volume fraction. This study aimed to confirm the synergistic anti-inflammatory and osteogenic preclinical effects of this system. The targets and pathways of parthenolide and naringin were predicted. Three cell models were used to assess the anti-inflammatory effects of parthenolide and the osteogenic effects of naringin. First, the distance between the cementoenamel junction and alveolar bone crest (CEJ-ABC) and the bone mineral density (BMD) of surgical defects were measured in a rat model of periodontitis with periodontal fenestration defects. Additionally, the mRNA expression levels of matrix metallopeptidase 9 (MMP9) and alkaline phosphatase (ALP) were measured. Furthermore, the number of inflammatory cells and osteoclasts, as well as the protein expression levels of tumor necrosis factor-alpha (TNF-α) and levels of ALP were determined. Target prediction suggested prostaglandin peroxidase synthase (PTGS2) as a potential target of parthenolide, while cytochrome P450 family 19 subfamily A1 (CYP19A1) and taste 2 receptor member 31 (TAS2R31) were potential targets of naringin. Parthenolide mainly targeted inflammation-related pathways, while naringin participated in steroid hormone synthesis and taste transduction. In vitro experiments revealed significant anti-inflammatory effects of parthenolide on RAW264.7 cells, and significant osteogenic effects of naringin on bone marrow mesenchymal stem cells and MC3T3-E1 cells. DDDS loaded with parthenolide and naringin decreased the CEJ-ABC distance and increased BMD and ALP levels in a time-dependent manner. Inflammation was significantly alleviated after 14 days of DDDS treatment. Additionally, after 56 days, the DDDS group exhibited the highest BMD and ALP levels. DDDS loaded with parthenolide and naringin in a rat model achieved significant synergistic anti-inflammatory and osteogenic effects, providing powerful preclinical evidence.

Clumps of mesenchymal stem cells/extracellular matrix complexes directly reconstruct the functional periodontal tissue in a rat periodontal defect model.

Periodontitis is an inflammatory disease characterized by tooth-supporting periodontal tissue destruction, including the cementum, periodontal ligament, and alveolar bone. To regenerate the damaged periodontal tissue, mesenchymal stem cells (MSCs) have attracted much scientific and medical attention. Recently, we generated clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs), which consist of cells and self-produced ECM. C-MSCs can be transplanted into lesion areas without artificial scaffold to induce tissue regeneration. To develop reliable scaffold-free periodontal tissue regenerative cell therapy by C-MSCs, this study investigated the periodontal tissue regenerative capacity of C-MSCs and the behavior of the transplanted cells. Rat bone marrow-derived MSCs were isolated from rat femur. Confluent cells were scratched using a micropipette tip and then torn off. The sheet was rolled to make a three-dimensional round clump of cells, C-MSCs. Then, ten C-MSCs were grafted into a rat periodontal fenestration defect model. To trace the grafted cells in the defect, PKH26-labeled cells were also employed. Micro-CT and histological analyses demonstrated that transplantation of C-MSCs induced successful periodontal tissue regeneration in the rat periodontal defect model. Interestingly, the majority of the cells in the reconstructed tissue, including cementum, periodontal ligaments, and alveolar bone, were PKH26 positive donor cells, suggesting the direct tissue formation by MSCs. This study demonstrates a promising scaffold-free MSCs transplantation strategy for periodontal disease using C-MSCs and offers the significance of multipotency of MSCs to induce successful periodontal tissue regeneration.

Ultrathin 2D Titanium Carbide MXene (Ti3 C2 Tx ) Nanoflakes Activate WNT/HIF-1α-Mediated Metabolism Reprogramming for Periodontal Regeneration.

Periodontal defect regeneration in severe periodontitis relies on the differentiation and proliferation of periodontal ligament cells (PDLCs). Recently, an emerging 2D nanomaterial, MXene (Ti3 C2 Tx ), has gained more and more attention due to the extensive antibacterial and anticancer activity, while its potential biomedical application on tissue regeneration remains unclear. Through a combination of experimental and multiscale simulation schemes, Ti3 C2 Tx has exhibited satisfactory biocompatibility and induced distinguish osteogenic differentiation of human PDLCs (hPDLCs), with upregulated osteogenesis-related genes. Ti3 C2 Tx manages to activate the Wnt/β-catenin signaling pathway by enhancing the Wnt-Frizzled complex binding, thus stabilizing HIF-1α and altering metabolic reprogramming into glycolysis. In vivo, hPDLCs pretreated by Ti3 C2 Tx display excellent performance in new bone formation and osteoclast inhibition with enhanced RUNX2, HIF-1α, and β-catenin in an experimental rat model of periodontal fenestration defects, indicating that this material has high efficiency of periodontal regeneration promotion. It is demonstrated in this work that Ti3 C2 Tx has highly efficient therapeutic effects in osteogenic differentiation and periodontal defect repairment.

Regeneration of rat periodontium by cementum protein 1-derived peptide.

Cementum protein 1 (CEMP1) has the capacity to promote differentiation of periodontal ligament (PDL) cells toward a cementoblastic phenotype in vitro and bone regeneration in vivo. In this study, we tested the capabilities of a synthetic cementum protein 1-derived peptide, MGTSSTDSQQAGHRRCSTSN (CEMP1-p1), to promote regeneration of periodontal structures in a periodontal fenestration defect in rats. Fenestration defects were created using an extra-oral approach in the buccal aspect of the mandibular first molar roots. Eighteen male Wistar rats were divided into three groups. Two controls (defects non-treated or defects treated with a gelatin matrix scaffold [GMS] only) and the experimental group treated with 5µg/dose of CEMP1-p1 embedded in GMS. After 28days, the animals were sacrificed, and the mandibles processed for histopathological examination. Expression of cementum proteins, cementum attachment protein (CAP), CEMP1, integrin binding sialoprotein (IBSP), and osteocalcin (OCN), was assessed using immunofluorescence. The formation of new cementum, bone, and PDL fibers were compared between control and experimental groups. The histological analysis revealed that the control group without any treatment new cementum or oriented PDL fibers were not observed. However, the presence of newly bone was detected. In the control group treated with GMS, new cementum formation was not detectable, the PDL fibers were oriented parallel to the longitudinal root axis, and new bone formation was observed. The experimental group showed deposit of acellular extrinsic fiber cementum (AEFC) in a lamellae-like feature with inserted Sharpey's fibers, formation of cellular mixed stratified cementum (CMSC) with the presence of cementocytes, and newly formed bone close to the cementum-enamel junction. Cementoblast cells adjacent to new cementum expressed CAP, CEMP1, IBSP, and OCN. These studies show that CEMP1-p1 promotes the formation of AEFC, CMSC, new PDL with Sharpey's fibers inserted in cementum and bone, thus providing strong evidence that the synthetic peptide CEMP1-p1 promotes periodontal regeneration in a rat fenestration model.

A Highly Ordered, Nanostructured Fluorinated CaP-Coated Melt Electrowritten Scaffold for Periodontal Tissue Regeneration.

Periodontitis is a chronic inflammatory, bacteria-triggered disorder affecting nearly half of American adults. Although some level of tissue regeneration is realized, its low success in complex cases demands superior strategies to amplify regenerative capacity. Herein, highly ordered scaffolds are engineered via Melt ElectroWriting (MEW), and the effects of strand spacing, as well as the presence of a nanostructured fluorinated calcium phosphate (F/CaP) coating on the adhesion/proliferation, and osteogenic differentiation of human-derived periodontal ligament stem cells, are investigated. Upon initial cell-scaffold interaction screening aimed at defining the most suitable design, MEW poly(ε-caprolactone) scaffolds with 500µm strand spacing are chosen. Following an alkali treatment, scaffolds are immersed in a pre-established solution to allow for coating formation. The presence of a nanostructured F/CaP coating leads to a marked upregulation of osteogenic genes and attenuated bacterial growth. In vivo findings confirm that the F/CaP-coated scaffolds are biocompatible and lead to periodontal regeneration when implanted in a rat mandibular periodontal fenestration defect model. In aggregate, it is considered that this work can contribute to the development of personalized scaffolds capable of enabling tissue-specific differentiation of progenitor cells, and thus guide simultaneous and coordinated regeneration of soft and hard periodontal tissues, while providing antimicrobial protection.

Inhibiting PHD2 in human periodontal ligament cells via lentiviral vector-mediated RNA interference facilitates cell osteogenic differentiation and periodontal repair.

Periodontal defect regeneration in severe periodontitis remains a challenging task in clinic owing to poor survival of seed cells caused by the remaining oxidative stress microenvironment. Recently, the reduction of prolyl hydroxylase domain-containing protein 2 (PHD2), a primary cellular oxygen sensor, has shown an incredible extensive effect on skeletal muscle tissue regeneration by improving cell resistance to reactive oxygen species, whereas its role in periodontal defect repair is unclear. Here, through lentivirus vector-mediated RNA interference, the PHD2 gene in human periodontal ligament cells (hPDLCs) is silenced, leading to hypoxia-inducible factor-1α stabilization in normoxia. In vitro, PHD2 silencing not only exhibited a satisfactory effect on cell proliferation, but also induced distinguished osteogenic differentiation of hPDLCs. Real-time polymerase chain reaction and Western blotting revealed significant up-regulation of osteocalcin, alkaline phosphatase (ALP), runt-related transcription factor 2, and collagen type I (COL I). Under oxidative stress conditions, COL I and ALP expression levels, suppressed by 100 μM H2 O2 , were elevated by PHD2-gene-silencing in hPDLCs. In vivo, periodontal fenestration defects were established in 18 female Sprague-Dawley rats aged 6wk old, followed by implantation of PHD2 silencing hPDLCs in situ for 21 d. Persistent and stable silencing of PHD2 in hPDLCs promoted better new bone formation according to microcomputed tomography 3D reconstruction and related bone parameter analysis. This work demonstrates the therapeutic efficiency of PHD2 gene interference in osteogenic differentiation and periodontal defect repair for highly efficient periodontal regeneration.

Inhibiting PHD2 in bone marrow mesenchymal stem cells via lentiviral vector-mediated RNA interference facilitates the repair of periodontal tissue defects in SD rats.

Hypoxia-inducible factors (HIFs) play an important role in angiogenesis, and they can activate the expression of several downstream angiogenic factors. HIF-1 is a major transcriptor of HIFs, composed of α and β subunits. Prolyl hydroxylase domain-containing protein 2 (PHD2) is the main catabolic enzyme for HIF-1α, and it can accelerate its degradation under normoxic conditions. PHD2 expression in bone marrow mesenchymal stem cells (BMMSCs) of SD rats was down-regulated under normoxic conditions in this study by utilizing lentiviral vector-mediated RNA interference to promote HIF-1α accumulation, thus enhancing the expression of angiogenic factors. A tissue-engineered compound was constructed using the composite collagen membrane of BMMSCs after PHD2 gene silencing to repair periodontal fenestration defects in SD rats. The results of this study indicated that, after PHD2 gene silencing, the osteogenic differentiation of BMMSCs was enhanced in vitro, the resistance of cells to oxidative stress was also validated in vitro, thereby illustrating the promotion of the repair of artificially constructed periodontal tissue defects in rats. The results of this study provide a reference and guidance for future applications of RNA interference in periodontal tissue engineering and serve as a basis for improving the survival of seed cells in recipient tissues.

Leptin Overexpression in Bone Marrow Stromal Cells Promotes Periodontal Regeneration in a Rat Model of Osteoporosis.

Osteoporosis is associated with widespread periodontitis and impaired periodontal healing. However, there is a lack of information about the outcomes of regenerative approaches under the influence of osteoporosis. This study investigates the effect of leptin (LEP) overexpression on the regenerative potential of bone marrow stromal cells (BMSCs) in an osteoporotic rat periodontal fenestration defect model. Rat BMSCs were transfected with adenoviruses harboring the human (h)LEP gene. Cell proliferation and osteogenic differentiation were evaluated. A β-tricalcium phosphate scaffold seeded with transfected cells was implanted into nude mice to investigate ectopic osteogenesis and into an osteoporotic rat defect to study periodontal regeneration. Regenerated periodontal and bone-like tissues were analyzed by histologic methods. hLEP overexpression induced osteogenic differentiation of BMSCs as evidenced by the upregulation of osteogenesis-related genes such as Runt-related transcription factor 2, alkaline phosphatase (ALP), and collagen Type I, as well as increased ALP activity and enhanced mineralization. Mice implanted with hLEP-BMSC-containing scaffolds showed more extensive formation of bone-like tissue than those in other groups. Periodontal defects were also filled to a greater degree when treated with hLEP-BMSCs and contained cementum and a well-organized periodontal ligament after 10 and 28 days. hLEP overexpression in BMSCs can stimulate periodontal regeneration in osteoporotic conditions and might be a promising strategy for periodontal regeneration in patients with osteoporosis.

Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration.

Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration.

Platelet-rich plasma and autogenous bone graft combined with guided tissue regeneration in periodontal fenestration defects in dogs.

The aim of this study was to evaluate the effects of platelet-rich plasma (PRP), autogenous bone (AB), and guided tissue regeneration (GTR) combination therapy compared to GTR therapy alone on healing of bone and cementum in fenestration-type periodontal defects in dogs. Six dogs were included in this study. Fenestration-type defects were created, and the following treatment groups were established: a control group treated with GTR alone and experimental groups treated with a combination of GTR + PRP, GTR + AB, and GTR + AB + PRP. The defects were evaluated by stereologic method and histomorphometric analysis, which were performed 4, 8, and 12 weeks postoperatively. The results showed a significant increase in trabecular bone area in the GTR + PRP group as compared with the control at 4 and 8 weeks (P < .05). The GTR + AB + PRP group showed significantly more trabecular bone area than both GTR and GTR + PRP groups at all time intervals (P < .05). The 8- and 12-week results in terms of cementum area revealed a significant difference between the GTR + AB + PRP group and the control in favor of the former (P < .05). Cementum area in the GTR + AB group was significantly greater than that in the GTR group at all time intervals (P < .05). Within the limitations of this study, PRP and AB, when used under barrier membrane, resulted in significant improvement in bone and cementum formation compared to GTR alone in periodontal fenestration defects; AB, rather than PRP, was responsible for this outcome.

Periodontal regeneration using strontium-loaded mesoporous bioactive glass scaffolds in osteoporotic rats.

Recent studies demonstrate that the rate of periodontal breakdown significantly increased in patients compromised from both periodontal disease and osteoporosis. One pharmacological agent used for their treatment is strontium renalate due to its simultaneous ability to increase bone formation and halt bone resorption. The aim of the present study was to achieve periodontal regeneration of strontium-incorporated mesoporous bioactive glass (Sr-MBG) scaffolds in an osteoporotic animal model carried out by bilateral ovariectomy (OVX). 15 female Wistar rats were randomly assigned to three groups: control unfilled periodontal defects, 2) MBG alone and 3) Sr-MBG scaffolds. 10 weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar and assessed by micro-CT and histomorphometric analysis after 28 days. Periodontal fenestration defects treated with Sr-MBG scaffolds showed greater new bone formation (46.67%) when compared to MBG scaffolds (39.33%) and control unfilled samples (17.50%). The number of TRAP-positive osteoclasts was also significantly reduced in defects receiving Sr-MBG scaffolds. The results from the present study suggest that Sr-MBG scaffolds may provide greater periondontal regeneration. Clinical studies are required to fully characterize the possible beneficial effect of Sr-releasing scaffolds for patients suffering from a combination of both periodontal disease and osteoporosis.

Platelet‐Rich Plasma, Low‐Level Laser Therapy, or Their Combination Promotes Periodontal Regeneration in Fenestration Defects: A Preliminary In Vivo Study

This study histomorphometrically analyzes the influence of platelet-rich plasma (PRP), low-level laser therapy (LLLT), or their combination on the healing of periodontal fenestration defects (PFDs) in rats. PFDs were surgically created in the mandibles of 80 rats. The animals were randomly divided into four groups: 1) C (control) and 2) PRP, defects were filled with blood clot or PRP, respectively; 3) LLLT and 4) PRP/LLLT, defects received laser irradiation, were filled with blood clot or PRP, respectively, and then irradiated again. Animals were euthanized at either 10 or 30 days post-surgery. Percentage of new bone (NB), density of newly formed bone (DNB), new cementum (NC), and extension of remaining defect (ERD) were histomorphometrically evaluated. Data were statistically analyzed (analysis of variance; Tukey test, P <0.05). At 10 days, group PRP presented ERD significantly lower than group C. At 30 days, group PRP presented NB and DNB significantly greater than group C. Groups LLLT, PRP, and PRP/LLLT showed significant NC formation at 30 days, with collagen fibers inserted obliquely or perpendicularly to the root surface. NC formation was not observed in any group C specimen. LLLT, PRP, or their combination all promoted NC formation with a functional periodontal ligament. The combination PRP/LLLT did not show additional positive effects compared to the use of either therapy alone.

Periodontal tissue regeneration by transplantation of rat adipose-derived stromal cells in combination with PLGA-based solid scaffolds.

Regeneration of damaged periodontium is challenging due to its multi-tissue composition. Mesenchymalstem cell-based approaches using adipose-derived stromal cells (ASCs) may contribute to periodontal reconstruction, particularly when combined with the use of scaffolds to maintain a space for new tissue growth. The aim of this study was to assess the regenerative potential of ASCs derived from inbred or outbred rats in combination with novel solid scaffolds composed of PLGA (Poly D,L-lactic-co-glycolic acid) (PLGA-scaffolds). Cultured ASCs seeded onto PLGA scaffolds (ASCs/PLGA) or PLGA-scaffolds (PLGA) alone were transplanted into periodontal fenestration defects created in F344 or Sprague Dawley (SD) rats. Micro-CT analysis showed a significantly higher percentage of bone growth in the ASCs/PLGA groups compared with the PLGA-alone groups at five weeks after surgery. Similarly, histomorphometric analysis demonstrated thicker growth of periodontal ligament and cementum layers in the ASCs/PLGA-groups compared with the PLGA-alone groups. In addition, transplanted DiI-labeled ASCs were observed in the periodontal regenerative sites. The present investigation demonstrated the marked ability of ASCs in combination with PLGA scaffolds to repair periodontal defects.

Assessment of the regenerative potential of allogeneic periodontal ligament stem cells in a rodent periodontal defect model

The complex microenvironment of the periodontal wound creates many challenges associated with multitissue regeneration of periodontal lesions. Recent characterization of mesenchymal stem cell-like populations residing in periodontal ligament tissues has shown that these cells exhibit features of postnatal stem cells. Despite these advances, a lack of consistency in design of preclinical studies and a limited study of allogeneic transplantation applications has restricted our understanding of their clinical utility in the treatment of periodontal disease. The aim of this study was to assess the regenerative potential of allogeneic periodontal ligament stem cells (PDLSCs) in a rat periodontal fenestration defect mode and to identify an optimal end time-point suitable for quantitative assessment of tissue regeneration. Periodontal fenestration defects, created in Sprague Dawley rats, were treated with allogeneic PDLSCs seeded onto Gelfoam(®) (Absorbable gelatin sponge; Pharmacia Corporation, Kalamazoo, MI, USA) or with Gelfoam(®) alone, or remained untreated. Experimental rats were killed at 7, 14, 21 or 28 d after surgery and the tissues were processed for immunohistochemical and histomorphometric examination. Defects treated with PDLSCs showed significantly greater percentage bone fill and length of new bone bridge compared with the untreated group or the group treated with Gelfoam(®) alone on days 14 and 21. Similarly, a statistically significant difference was achieved within specimens retrieved on day 21 for analysis of regeneration of cementum/periodontal ligament (PDL)-like structures. The present investigation shows that allogeneic PDLSCs have a marked ability to repair periodontal defects by forming bone, PDL and cementum-like tissue in vivo. The results suggest that treatment periods of 14 and 21 d are optimal end time-points for quantitative assessment of periodontal regeneration within the rodent fenestration-defect model utilized in the present study.

Effects of bone morphogenetic protein‐6 on periodontal wound healing/regeneration in supraalveolar periodontal defects in dogs

Application of a synthetic BMP-6 polypeptide in a rat periodontal fenestration defect model enhanced periodontal wound healing/regeneration including new bone and cementum formation. The purpose of this study was to translate the relevance of these initial observations into a discriminating large animal model. Critical-size (4-5 mm) supraalveolar periodontal defects were created at the 2(nd) and 3(rd) mandibular premolar teeth in 11 Beagle dogs. Experimental sites received BMP-6 at 0.25, 1.0 and 2.0 mg/ml soak-loaded onto an absorbable collagen sponge (ACS) carrier or ACS alone (control) each condition repeated in four jaw quadrants. The animals were euthanized at 8 weeks when block biopsies were collected and processed for histologic/histometric analysis. BMP-6 at 0.25, 1.0 and 2.0 mg/ml soak-loaded onto the ACS yielded significantly enhanced new bone (0.99 ± 0.07 versus 0.23 ± 0.13 mm/BMP-6 at 0.25 mg/ml) and cementum (2.45 ± 0.54 versus 0.73 ± 0.15 mm/BMP-6 at 0.25 mg/ml) formation including a functionally oriented periodontal ligament compared with control (p < 0.05). A significant inverse linear association between BMP-6 dose and new bone (β = -0.21 ± 0.09 mm, p = 0.016) and cementum height (β = -0.34 ± 0.15 mm, p = 0.023) was observed. Minimal root resorption was observed without significant differences between groups. Ankylosis was not observed for any of the experimental groups. Surgical application of BMP-6/ACS onto critical-size supraalveolar defects enhanced periodontal wound healing/regeneration, in particular cementogenesis including a functionally oriented periodontal ligament; the low BMP-6 0.25 mg/ml concentration apparently providing the most effective dose.

Application of eGFP to label human periodontal ligament stem cells in periodontal tissue engineering

ObjectivesTo establish human periodontal ligament stem cells (hPDLSC) with high and stable expression of enhanced green fluorescent protein (eGFP) and to obtain an ideal vector expression system that suitable for gene therapy in periodontal tissue engineering. MethodshPDLSCs were transfected with eGFP for 48h via different MOI (25, 50, 100, 200 and 400) by lentiviral vector, the transfection efficiency was evaluated by fluorescent microscopy and flow cytometry, and transfected hPDLSCs proliferation was evaluated by MTT. Pluripotent, differentiation capacity and ALP expression status were determined further. Osteoblast-associated genes expressions for osteogenesis were evaluated by quantitative-PCR. In addition, rat molar periodontal fenestration defect model was used for evaluating periodontal tissue engineering. ResultsThe transfection efficiency after 48h were 44.7%, 60.9%, 71.7%, 85.8%, and 86.9% respectively. There was no significant effect of transfection (at different MOI levels of 25, 50, 100, and 200) on the proliferation of hPDLSCs (designated as eGFP-hPDLSCs) compared with hPDLSCs (P>0.05). However, proliferation of eGFP hPDLSCs at MOI 400 became slower (P<0.05). Both eGFP hPDLSCs and hPDLSCs were able to differentiate into osteocytes and adipocytes under certain conditioned media. At 7 days, expression levels of COL-1, RUNX2 in hPDLSCS were higher than those in eGFP hPDLSCs (P<0.05); expression levels of ALP and OPN in eGFP hPDLSCs were similar to those in hPDLSCs (P>0.05). Newly regenerated bone formation was observed in the defect model used. ConclusionsAmong the transfection conditions, 48h transfection at MOI 200 is optimal for labelling hPDLSCs with eGFP in a lentiviral vector. There is no change in capability of the eGFP hPDLSCs osteogenesis. The lentiviral vector with eGFP is an appropriate expression vector system and hPDLSCs are ideal seeding cells for gene therapy in periodontal tissue engineering.

Effect of two delivery systems for recombinant human bone morphogenetic protein-2 on periodontal regeneration in vivo

Resorbable collagen membranes for guided tissue regeneration in periodontal therapy have shown promise but are not osteoinductive. As recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to have an affinity for collagen, the use of this osteoinductive agent incorporated into a collagen vehicle may act as a suitable carrier to promote periodontal regeneration. The aim of this study was to investigate the effects of two different collagen delivery systems for rhBMP-2 in rat periodontal fenestration defects. Using the collagen membrane delivery system, 3 groups of adult Wistar rats which had surgical defects created on the right side of the mandible involving the removal of bone and exposure of the molar roots were treated with either rhBMP-2 in collagen membrane (BMPm) (n= 12 animals), or collagen membrane only (COLm) (n = 12), or were left untreated (UN) (n= 14). Using the collagen gel delivery system, surgical defects were treated with either rhBMP-2 incorporated in a collagen gel carrier (BMPg) (n = 5) or had collagen gel only (COLg) (n = 6). Animals were killed 10 d postoperatively and tissues processed for histology. New bone formation was significantly greater in BMPg compared with both BMPm and controls (p<0.05). However, new cementum formation was significantly greater in BMPm (721 ± 166 μm2, mean ± SE) compared with COLm, COLg and UN (p<0.02) (190±44μm2, 327±114 μm2 and 172 ± 33 μm2, respectively) and more than 1.5 times BMPg (451 ± 158 μm2). In conclusion, both carrier systems for rhBMP-2 significantly increased new bone formation compared with controls during the early stages of periodontal wound healing. However, the more slowly dissolving collagen membrane carrier system for rhBMP-2 produced significantly greater new cementum compared with the collagen gel carrier, suggesting that a more prolonged exposure of rhBMP-2 is required to increase cementogenesis.