Multicompartmental Scaffolds for Coordinated Periodontal Tissue Engineering.

Successful periodontal repair and regeneration requires the coordinated responses from soft and hard tissues as well as the soft tissue-to-bone interfaces. Inspired by the hierarchical structure of native periodontal tissues, tissue engineering technology provides unique opportunities to coordinate multiple cell types into scaffolds that mimic the natural periodontal structure in vitro. In this study, we designed and fabricated highly ordered multicompartmental scaffolds by melt electrowriting, an advanced 3-dimensional (3D) printing technique. This strategy attempted to mimic the characteristic periodontal microenvironment through multicompartmental constructs comprising 3 tissue-specific regions: 1) a bone compartment with dense mesh structure, 2) a ligament compartment mimicking the highly aligned periodontal ligaments (PDLs), and 3) a transition region that bridges the bone and ligament, a critical feature that differentiates this system from mono- or bicompartmental alternatives. The multicompartmental constructs successfully achieved coordinated proliferation and differentiation of multiple cell types in vitro within short time, including both ligamentous- and bone-derived cells. Long-term 3D coculture of primary human osteoblasts and PDL fibroblasts led to a mineral gradient from calcified to uncalcified regions with PDL-like insertions within the transition region, an effect that is challenging to achieve with mono- or bicompartmental platforms. This process effectively recapitulates the key feature of interfacial tissues in periodontium. Collectively, this tissue-engineered approach offers a fundament for engineering periodontal tissue constructs with characteristic 3D microenvironments similar to native tissues. This multicompartmental 3D printing approach is also highly compatible with the design of next-generation scaffolds, with both highly adjustable compartmentalization properties and patient-specific shapes, for multitissue engineering in complex periodontal defects.

Maresin 1 Promotes Wound Healing and Socket Bone Regeneration for Alveolar Ridge Preservation.

Tooth extraction results in alveolar bone resorption and is accompanied by postoperative swelling and pain. Maresin 1 (MaR1) is a proresolving lipid mediator produced by macrophages during the resolution phase of inflammation, bridging healing and tissue regeneration. The aim of this study was to examine the effects of MaR1 on tooth extraction socket wound healing in a preclinical rat model. The maxillary right first molars of Sprague-Dawley rats were extracted, and gelatin scaffolds were placed into the sockets with or without MaR1. Topical application was also given twice a week until complete socket wound closure up to 14 d. Immediate postoperative pain was assessed by 3 scores. Histology and microcomputed tomography were used to assess socket bone fill and alveolar ridge dimensional changes at selected dates. The assessments of coded specimens were performed by masked, calibrated examiners. Local application of MaR1 potently accelerated extraction socket healing. Macroscopic and histologic analysis revealed a reduced soft tissue wound opening and more rapid re-epithelialization with MaR1 delivery versus vehicle on socket healing. Under micro-computed tomography analysis, MaR1 (especially at 0.05 µg/µL) stimulated greater socket bone fill at day 10 as compared with the vehicle-treated animals, resulting in less buccal plate resorption and a wider alveolar ridge by day 21. Interestingly, an increased ratio of CD206+:CD68+ macrophages was identified in the sockets with MaR1 application under immunohistochemistry and immunofluorescence analysis. As compared with the vehicle therapy, local delivery of MaR1 reduced immediate postoperative surrogate pain score panels. In summary, MaR1 accelerated extraction wound healing, promoted socket bone fill, preserved alveolar ridge bone, and reduced postoperative pain in vivo with a rodent preclinical model. Local administration of MaR1 offers clinical potential to accelerate extraction socket wound healing for more predictable dental implant reconstruction.

Effect of sustained PDGF nonviral gene delivery on repair of tooth-supporting bone defects.

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) promotes soft tissue and bone healing, and is Food and Drug Administration-approved for treatment of diabetic ulcers and periodontal defects. The short half-life of topical rhPDGF-BB protein application necessitates bolus, high-dose delivery. Gene therapy enables sustained local growth factor production. A novel gene activated matrix delivering polyplexes of polyethylenimine (PEI)-plasmid DNA encoding PDGF was evaluated for promotion of periodontal wound repair in vivo. PEI-pPDGF-B polyplexes were tested in human periodontal ligament fibroblasts and human gingival fibroblasts for cell viability and transfection efficiency. Collagen scaffolds containing PEI-pPDGF-B polyplexes at two doses, rhPDGF-BB, PEI vector or collagen alone were randomly delivered to experimentally induced tooth-supporting periodontal defects in a rodent model. Mandibulae were collected at 21 days for histologic observation and histomorphometry. PEI-pPDGF-B polyplexes were biocompatible to cells tested and enzyme-linked immunosorbent assay confirmed the functionality of transfection. Significantly greater osteogenesis was observed for collagen alone and rhPDGF-BB versus the PEI-containing groups. Defects treated with sustained PDGF gene delivery demonstrated delayed healing coupled with sustained inflammatory cell infiltrates lateral to the osseous defects. Continuous PDGF-BB production by nonviral gene therapy could have delayed bone healing. This nonviral gene delivery system in this model appeared to prolong inflammatory response, slowing alveolar bone regeneration in vivo.

LIM domain protein-3 (LMP3) cooperates with BMP7 to promote tissue regeneration by ligament progenitor cells.

Gene transfer of key regulators of osteogenesis for mesenchymal stem cells represents a promising strategy to regenerate bone. It has been reported that LMP3, a transcription variant of LIM domain mineralization protein (LMP) lacking LIM domains, can induce osteogenesis in vitro and in vivo. As little is known about the effects of LMP3 gene therapy on periodontal ligament (PDL) cell osteogenic differentiation, this study sought to explore whether gene delivery of LMP3 can promote PDL cell mineralization and bone formation. Our results showed that adenoviral mediated gene transfer of LMP3 (AdLMP3) significantly upregulated ALP (Alkaline Phosphatase), BSP (Bone Sialoprotein) and BMP2 gene expression and increased in vitro matrix mineralization in human PDL. Although AdLMP3 gene delivery to PDL cells did not induce ectopic bone formation in vivo, we found that AdLMP3 augments new bone formation, which co-delivered with AdBMP7 gene transfer. Our study provides the evidence that there is a synergistic effect between LMP3 and BMP-7 in vivo, suggesting that LMP3 delivery may be used to augment BMP-mediated osteogenesis. LMP3 and BMP-7 combinatory gene therapy may also have specific applications for oral and periodontal regenerative medicine.

Saliva/pathogen biomarker signatures and periodontal disease progression.

The purpose of this study was to determine the role of saliva-derived biomarkers and periodontal pathogens during periodontal disease progression (PDP). One hundred human participants were recruited into a 12-month investigation. They were seen bi-monthly for saliva and clinical measures and bi-annually for subtraction radiography, serum and plaque biofilm assessments. Saliva and serum were analyzed with protein arrays for 14 pro-inflammatory and bone turnover markers, while qPCR was used for detection of biofilm. A hierarchical clustering algorithm was used to group study participants based on clinical, microbiological, salivary/serum biomarkers, and PDP. Eighty-three individuals completed the six-month monitoring phase, with 39 [corrected] exhibiting PDP, while 44 [corrected] demonstrated stability. Participants assembled into three clusters based on periodontal pathogens, serum and salivary biomarkers. Cluster 1 members displayed high salivary biomarkers and biofilm; 71% [corrected] of these individuals were undergoing PDP. Cluster 2 members displayed low biofilm and biomarker levels; 76% [corrected] of these individuals were stable. Cluster 3 members were not discriminated by PDP status; however, cluster stratification followed groups 1 and 2 based on thresholds of salivary biomarkers and biofilm pathogens. The association of cluster membership to PDP was highly significant (p < 0.0007). [corrected] The use of salivary and biofilm biomarkers offers potential for the identification of PDP or stability (ClinicalTrials.gov number, CT00277745).

Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction.

BACKGROUND AND OBJECTIVE: Platelet-derived growth factor-BB is a potent mediator of tooth-supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet-derived growth factor-BB application is its short half-life in vivo. Gene therapy has shown strong promise for the long-term delivery of platelet-derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet-derived growth factor-B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad-PDGF-B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell-surface proteins associated with cellular signaling. MATERIAL AND METHODS: HGFs from human subjects were treated by adenoviral PDGF-B, PDGF-1308 (a dominant negative mutant of PDGF) and recombinant human platelet-derived growth factor-BB, and then incubated in serum-free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF-B was measured by RT-PCR and Western blot. Cell proliferation was evaluated by [methyl-3H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF-B gene transfer. The expression of alpha and beta PDGFR and Akt were measured by Western blot analysis. RESULTS: Sustained in vitro expression of PDGF-B in HGFs by Ad-PDGF-B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF-BB and Ad-PDGF-1308, Ad-PDGF-B maintained cell growth in serum-free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF-B also prolonged downregulation of the growth arrest specific gene (gas) PDGF alpha R. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long-term cell signaling effect as a result of the over-expression of Ad-PDGF-B, compared with pulse recombinant human platelet-derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad-PDGF-B, for at least up to 96 h. CONCLUSION: These findings further demonstrate that gene delivery of PDGF-B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet-derived growth factor-BB protein. These data on platelet-derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.

Current concepts in periodontal bioengineering.

Repair of tooth supporting alveolar bone defects caused by periodontal and peri-implant tissue destruction is a major goal of reconstructive therapy. Oral and craniofacial tissue engineering has been achieved with limited success by the utilization of a variety of approaches such as cell-occlusive barrier membranes, bone substitutes and autogenous block grafting techniques. Signaling molecules such as growth factors have been used to restore lost tooth support because of damage by periodontal disease or trauma. This paper will review emerging periodontal therapies in the areas of materials science, growth factor biology and cell/gene therapy. Several different polymer delivery systems that aid in the targeting of proteins, genes and cells to periodontal and peri-implant defects will be highlighted. Results from preclinical and clinical trials will be reviewed using the topical application of bone morphogenetic proteins (BMP-2 and BMP-7) and platelet-derived growth factor-BB (PDGF) for periodontal and peri-implant regeneration. The paper concludes with recent research on the use of ex vivo and in vivo gene delivery strategies via gene therapy vectors encoding growth promoting and inhibiting molecules (PDGF, BMP, noggin and others) to regenerate periodontal structures including bone, periodontal ligament and cementum.

Expression of collagens I, II, X, and XI and aggrecan mRNAs by bovine growth plate chondrocytes in situ.

The cells responsible for skeletal growth are the chondrocytes of the cartilaginous growth plate. These cells differentiate through a series of maturational stages, establishing different zones in the growth plate. Among the major functions of these cells is the production of appropriate extracellular matrix, primarily composed of collagens and proteoglycans. To determine whether matrix synthesis varies with respect to maturational stage and in which cell populations different collagens are expressed, bovine growth plates were analyzed by in situ hybridization to mRNA and by Northern blot hybridization. The most abundant collagen mRNA in the growth plate was type-II collagen. This mRNA was present at relatively low levels in the most immature cells of the growth plate but increased several-fold as cells entered the proliferative stage and remained high through subsequent phases of maturation. Type-XI collagen mRNA and mRNA for the cartilage-characteristic proteoglycan, aggrecan, were codistributed with the type-II collagen mRNA; however, both were present in much smaller quantities. Type-X procollagen mRNA was localized to chondrocytes late in their maturation and was expressed at levels similar to the expression of type-II collagen. In situ hybridization of serial sections revealed that growth plate chondrocytes in their more mature stages contain both type-II and type-X collagen mRNA. Type-I collagen mRNA was not observed in growth plate chondrocytes at any maturational stage; rather, it was localized to a morphologically distinct population of cells attached to calcifying cartilage septa in the region of vascular invasion.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis.

Monoclonal antibodies (MAb) 3F11 and 06B4 recognize epitopes that are conserved on gonococcal lipooligosaccharides (LOS), present on some meningococcal LOS, and conserved on human erythrocytes. LOS of some group B and C prototype meningococcal LOS strains (LOS serotypes L1 to L8) treated with neuraminidase showed increased expression of the 3F11 and 06B4 MAb-defined epitopes. Neuraminidase-treated LOS separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stained showed a shift in migration from a component with a mass of approximately 4.8 kDa to a component with a mass of between 4.5 and 4.6 kDa. The same strains grown in medium with excess CMP-N-acetylneuraminic acid had LOS that shifted in migration to a slightly higher component (mass, approximately 4.8 kDa). Chemical analysis of the neuraminidase-digested products from one LOS indicated it contained approximately 1.5% sialic acid. Covalent linkage between sialic acid and the LOS was confirmed by analysis of de-O-acylated and dephosphorylated LOS by liquid secondary ion mass spectrometry. Three studies show that some meningococci contain sialic acid in their LOS, that the sialic acid is cleaved and lost in conventional acetic acid hydrolysis, and that the sialic acid alters the expression of MAb-defined epitopes.

In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.