Periodontal Ligament Collagen Research Articles

Understanding the hierarchical structure of collagen fibers of the human periodontal ligament: Implications for biomechanical characteristics

The periodontal ligament (PDL) is a unique fibrous connective tissue that regulates periodontal homeostasis mechanisms. Its biomechanical properties primarily reside in the hierarchical and non-uniform collagenous network. This study aimed to investigate the region-specific structure and composition of collagen fibers in the PDL at various scales and to explore their relationship with mechanical properties in a split-mouth design. Fresh human cadaver transverse PDL specimens of maxillary anterior teeth were categorized into cervical, middle, and apical groups. These specimens were analyzed via Masson’s trichrome staining, scanning electron microscopy, picrosirius red (PSR) staining, three-dimensional (3D) reconstruction, Raman spectroscopy, and uniaxial tensile test. Statistical analyses were performed to compare the structural, compositional, and tensile properties among the groups. Notably, the middle PDL samples exhibited superior tensile strength and higher fiber area fraction than the other two transverse sections. Despite a higher mineral-to-matrix ratio and a different collagen secondary structure, the apical PDL demonstrated a relatively weaker tensile strength, possibly associated with its discovered sparser collagen fiber areal fraction. The cervical region, characterized by a mediocre fiber areal fraction, displayed diminished tensile strength. The 3D reconstructed collagenous network model and PSR staining exposed the fiber interaction and the micropores. Microscale porosity and variations in collagen secondary structure, particularly in the apical region, suggest adaptive mechanisms for accommodating compressive forces and maintaining functional integrity. Variance in the tensile properties of samples in different force directions indicated the significant influence of fiber orientation and root level on tissue mechanics. Statement of significanceThis study provides critical insights into the biomechanical and structural properties of the human periodontal ligament (PDL), particularly focusing on the underexplored anterior teeth. Through advanced techniques like SEM, histological staining, 3D reconstruction, Raman spectroscopy, and tensile testing, we reveal significant regional variations in PDL collagen organization, composition, and biomechanical properties. Our findings address a crucial knowledge gap concerning the material mechanics of the PDL, offering a foundational understanding for future periodontal tissue engineering and biomimetic material development. This multi-scale analysis underscores the importance of both mesoscale structural characteristics and nanoscale molecular structures in maintaining PDL mechanical integrity.

Calcium phosphate nanoclusters modify periodontium remodeling and minimize orthodontic relapse.

Orthodontic relapse is one of the most prevalent concerns of orthodontic therapy. Relapse results in patients' teeth reverting towards their pretreatment positions, which increases the susceptibility to functional problems, dental disease, and substantially increases the financial burden for retreatment. This phenomenon is thought to be induced by rapid remodeling of the periodontal ligament (PDL) in the early stages and poor bone quality in the later stages. Current therapies, including fixed or removable retainers and fiberotomies, have limitations with patient compliance and invasiveness. Approaches using biocompatible biomaterials, such as calcium phosphate polymer-induced liquid precursors (PILP), is an ideal translational approach for minimizing orthodontic relapse. Here, post-orthodontic relapse is reduced after a single injection of high concentration PILP (HC-PILP) nanoclusters by altering PDL remodeling in the early stage of relapse and improving trabecular bone quality in the later phase. HC-PILP nanoclusters are achieved by using high molecular weight poly aspartic acid (PASP, 14 kDa) and poly acrylic acid (PAA, 450 kDa), which resulted in a stable solution of high calcium and phosphate concentrations without premature precipitation. In vitro results show that HC-PILP nanoclusters prevented collagen type-I mineralization, which is essential for the tooth-periodontal ligament (PDL)-bone interphase. In vivo experiments show that the PILP nanoclusters minimize relapse and improve the trabecular bone quality in the late stages of relapse. Interestingly, PILP nanoclusters also altered the remodeling of the PDL collagen during the early stages of relapse. Further in vitro experiments showed that PILP nanoclusters alter the fibrillogenesis of collagen type-I by impacting the protein secondary structure. These findings propose a novel approach for treating orthodontic relapse and provide additional insight into the PILP nanocluster's structure and properties on collagenous structure repair.

MORPHOLOGICAL CHANGES OF RAT PERIODONTIUM IN SUPRAOCCLUSAL RELATIONS OF INDIVIDUAL TEETH IN THE AGE ASPECT

Although the issue of the occlusal load influence on the occurrence of pathological phenomena in the body has been discussed for decades, it is still a subject of debate. Pathogenic occlusion is defined as excessive occlusal forces (premature contacts and interference) that can cause injuries to the dentition, including teeth and periodontal tissues. Changes in occlusal dimensions, if not properly performed or treated, can lead to dysfunctions of the dentition, such as joint clicking, difficulty in chewing, occlusal discomfort, muscle fatigue, and pain.
 Aim: to determine the morphological changes of periodontium in rats of different age groups in the supraocclusal relationship of individual teeth in the age aspect.
 Materials and methods of the study. The study was conducted on 60 white laboratory rats aged 4 to 22 months and weighing 90 to 330 g. The rats were divided into control (30 animals) and experimental (30 animals) groups. Each group was divided into 3 subgroups (10 animals each) depending on the age of the rats: young, mature and senile. Young animals included rats aged 3 4 months with a weight of 90 100 g; mature animals aged 6-8 months with a weight of 150 170 g; senile rats 20 22 months and weighing 200 220 g. In the experimental group, the state of supraocclusion was modeled by increasing the height of the lower right second molars by placing 1 mm high filling. For the placement of the filling, 37% orthophosphorus etching gel for enamel and dentin (Latus, Ukraine), microapplicators (Latus, Ukraine), the fifth generation Prime&Bond NT adhesive system (Dentsply Sirona, Germany), the universal microhybrid light-curing composite material Latelux (Latus, Ukraine) and the photopolymerizer LED.D (WOODPECKER, China) were used according to the manufacturer's instructions. The experimental animals were withdrawn from the experiment on the 15th day by decapitation under general anesthesia with sodium thiopental. The lower jaws were skeletonized, and dental blocks were cut out in the area of the molars. For histological examination, the dental blocks were fixed in a 10% solution of neutral formalin for two days, dehydrated in alcohols of increasing concentration and embedded in paraffin. Then, sections were made from the obtained preparations on a Shandon Finesse 325 rotary microtome with a section thickness of 4-6 μm. The histological sections were fixed on degreased slides and dried. Van Gieson staining was performed. The width of the periodontal gap was determined using the ZEISS ZEN 3.7 program by Carl Zeiss Microscopy.
 Results of the study. Histometric analysis has showed significant morphological changes in the experimental group of all age categories. In young rats, the fibrinoid edema phenomena of the periodontal ligament collagen fibers were observed. In mature rats, there was a picture of mucoid edema of collagen fibers of the periodontal ligament. In senile rats, fibrinoid necrosis of collagen fibers of the periodontal ligament were discovered, an inflammatory infiltrate, macrophages and neutrophils, with a predominance of neutrophilic inflammatory microenvironment, and initial hyaline tissue changes were found. In all age groups, there was cement resorption on the lateral surfaces of the tooth root, lacunar resorption of alveolar bone tissue and the presence of a large number of osteoclasts. In senile rats, hypercementosis was determined in the apical part of the root. In the control group of all age categories, neither resorption nor disorganization of connective tissue, nor hypercementosis was detected.
 Conclusions. The study shows that traumatic occlusion leads to resorption of root and alveolar cementum in all age groups. In young rats, traumatic occlusion causes fibrinoid edema of collagen fibers of the periodontal ligament. In mature rats, traumatic occlusion causes mucoid edema of collagen fibers of the periodontal ligament. In senile rats, traumatic occlusion causes fibrinoid necrosis of collagen fibers of the periodontal ligament, initial hyaline tissue changes and hypercementosis.

An Immunohistochemical and Histological Study of the Animal Periodontal Ligament During Orthodontic Force Application with Concomitant Application of Electric Current - An Animal Study.

The application of direct current can have a significant impact on the rate of tooth movement and surrounding periodontal ligament collagen turnover. This study aims to provide insight into the optimal characteristics of applied current to achieve enhanced tissue response. Eighteen male Wistar rats were divided into three groups (I, II, and III). Split mouth design was used, and each side was allocated into an experimental group or control group. Experimental sides of groups I, II, and III received 20, 10, and 15 μA of current (15 min, twice daily for 3 days). Both the experimental and control groups receive an orthodontic force via the NiTi closed coil spring. The amount of tooth movement was determined daily. Immunohistochemistry slides were scored using the immunoreactive scoring (IRS) system for collagen types I and III. One-way Analysis of Variance (ANOVA) and Tukey post hoc test were used to analyse the rate of tooth movement, while Mann-Whitney test was used to analyse IRS distribution between control and experimental groups. Compared with the control group, there was a statistically significant difference in tooth movement in all the experimental groups, with group 3 showing the maximum rate on days 2 and 3. This was supported by immunoreactive scores for both collagen types I and III. After 72 hours, the expression of collagen types 1 and 3 increased significantly for group III. This finding was in harmony with the rate of tooth movement, which was maximum for group 3 (15 μA) as compared to other groups.

Mice Lacking PLAP-1/Asporin Show Alteration of Periodontal Ligament Structures and Acceleration of Bone Loss in Periodontitis.

Periodontal ligament-associated protein 1 (PLAP-1), also known as Asporin, is an extracellular matrix protein expressed in the periodontal ligament and plays a crucial role in periodontal tissue homeostasis. Our previous research demonstrated that PLAP-1 may inhibit TLR2/4-mediated inflammatory responses, thereby exerting a protective function against periodontitis. However, the precise roles of PLAP-1 in the periodontal ligament (PDL) and its relationship to periodontitis have not been fully explored. In this study, we employed PLAP-1 knockout mice to investigate its roles and contributions to PDL tissue and function in a ligature-induced periodontitis model. Mandibular bone samples were collected from 10-week-old male C57BL/6 (WT) and PLAP-1 knockout (KO) mice. These samples were analyzed through micro-computed tomography (μCT) scanning, hematoxylin and eosin (HE) staining, picrosirius red staining, and fluorescence immunostaining using antibodies targeting extracellular matrix proteins. Additionally, the structure of the PDL collagen fibrils was examined using transmission electron microscopy (TEM). We also conducted tooth extraction and ligature-induced periodontitis models using both wild-type and PLAP-1 KO mice. PLAP-1 KO mice did not exhibit any changes in alveolar bone resorption up to the age of 10 weeks, but they did display an enlarged PDL space, as confirmed by μCT and histological analyses. Fluorescence immunostaining revealed increased expression of extracellular matrix proteins, including Col3, BGN, and DCN, in the PDL tissues of PLAP-1 KO mice. TEM analysis demonstrated an increase in collagen diameter within the PDL of PLAP-1 KO mice. In line with these findings, the maximum stress required for tooth extraction was significantly lower in PLAP-1 KO mice in the tooth extraction model compared to WT mice (13.89 N ± 1.34 and 16.51 N ± 1.31, respectively). In the ligature-induced periodontitis model, PLAP-1 knockout resulted in highly severe alveolar bone resorption, with a higher number of collagen fiber bundle tears and significantly more osteoclasts in the periodontium. Our results demonstrate that mice lacking PLAP-1/Asporin show alteration of periodontal ligament structures and acceleration of bone loss in periodontitis. This underscores the significant role of PLAP-1 in maintaining collagen fibrils in the PDL and suggests the potential of PLAP-1 as a therapeutic target for periodontal diseases.

Construction of Human Periodontal Ligament Constitutive Model Based on Collagen Fiber Content.

Periodontal ligament (PDL) is mainly composed of collagen fiber bundles, and the content of collagen fiber is an important factor affecting the mechanical properties of PDL. Based on this, the purpose of this study is to explore the effect of the PDL collagen fiber content on its viscoelastic mechanical behavior. Transverse and longitudinal samples of different regions of PDL were obtained from the human maxilla. The fiber content at different regions of human PDL was quantitatively measured using image processing software, and a new viscoelastic constitutive model was constructed based on the fiber content. The nano-indentation experiment was carried out with a loading rate of 0.5 mN·s-1, a peak load of 3 mN, and a holding time of 200 s, and the model parameters were obtained through the experiment data. The results showed that with the increase of fiber content, the deformation resistance of PDL also increased, and compared with the neck and middle region, the compressive strain in the apical region of PDL was the largest. The range of reduced elastic modulus of human PDL was calculated to be 0.39~5.08 MPa. The results of the experimental data and the viscoelastic constitutive model fit well, indicating that the model can well describe the viscoelastic behavior of human PDL.

Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress.

The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.

Functional periodontal regeneration using biomineralized extracellular matrix/stem cell microspheroids

Engineering complete periodontal tissue regeneration remains a considerable clinical challenge because the native periodontium is composed of hard mineralized and soft unmineralized tissues. Stem cell therapies that use endogenous extracellular matrix (ECM) have the potential for structural and functional periodontal regeneration. Here, mineralized ECM/dental pulp stem cell microspheroids (MMCMs) mimicking native mesenchymal microspheroid precursors were developed, and showed an enhanced Young’s modulus ranging from 0.4 GPa to 1.9 GPa. Furthermore, biomineralization promoted cell viability, inhibited cell apoptosis, and enhanced ECM secretion and osteogenic differentiation potential in dental pulp stem cells. After implantation in the full periodontal defect, MMCMs regenerated complete periodontium with native-like hierarchically organized periodontal ligament collagen fibers inserted into the newly formed alveolar bone and cementum; they also recruited CD90+CD73+ host mesenchymal stem cells. Mechanistically, MMCMs promoted functional periodontal regeneration through activation of the transforming growth factor beta 1 (TGF-β1)/Smad3 signaling pathway. Inhibition of TGF-β1 signaling pathway significantly impaired osteogenic potential and matrix secretion of MMCMs, while exogenous TGF-β1 treatment enhanced the osteogenesis and periodontal regeneration outcomes of MMCMs. These results indicate the promising application of MMCMs in complex tissue regeneration.

Platelet-derived growth factor-BB regenerates functional periodontal ligament in the tooth replantation

Tooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rβ- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear β-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on β-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.

Histological, Histochemical and Immunohistochemical Evaluation of the Role of Bone Marrow-Derived Mesenchymal Stem Cells on the Structure of Periodontal Tissues in Carbimazole-Treated Albino Rats

ObjectiveTo elucidate the role of bone marrow-mesenchymal stem cells (BM-MSCs) on the structure of periodontal tissues in carbimazole (antithyroid drug) treated rats at different durations. Design28 albino rats were divided into: Group I: received distilled water. Group II: received therapeutic dose of carbimazole. Group III: received carbimazole then single injection of BM-MSCs by the end of 3rd week. Group IV: received carbimazole and single injection of BM-MSCs at the beginning of the experiment. Specimens were examined by light microscope. New collagen and β-catenin-immunoreactivity area% were assessed histomorphometrically, and statistically using ANOVA test. ResultsHistological examination revealed normal periodontal tissues structure in Groups I & IV. Group II showed disorganized periodontal ligament fibers and different stainability of cementum and alveolar bone. Group III illustrated dense periodontal ligament fibers, normal stainability of cementum and most of alveolar bone. Masson's trichrome results of Groups I & IV illustrated large areas of new collagen in periodontal ligament, old collagen in cementum and intermingled old and new collagen in alveolar bone. Group II showed old collagen. Group III revealed only new collagen. β-catenin-immunoreactivity was strong in Groups I & IV, negative in Group II and moderate in Group III. Statistically, Group III showed highest mean of new collagen area% followed by Groups I, IV and II respectively. Highest mean of β-catenin-immunoreactivity area% was for Group I followed by Groups IV, III and II respectively. ConclusionsCarbimazole has damaging effects and BM-MSCs are capable to mend these destructive outcomes in time dependent manner.

Three-dimensional ultrastructural imaging and quantitative analysis of the periodontal ligament.

The periodontal ligament (PDL) is a unique connective tissue mainly comprising collagen fiber bundles and cells between the roots of teeth and inner walls of the alveolar-bone socket. PDL fiber bundles are arrayed between teeth and bone, with both ends embedded in the cementum or alveolar bone as Sharpey's fiber. These bundles, synthesized by PDL fibroblasts (PDLFs), form several distinct groups within the PDL which has important functions besides tooth anchoring including tooth nutrition, proprioception, sensory detection, homoeostasis, and repair of damaged tissue. However, little is known about how the regular-PDL fiber bundle arrays are formed, maintained, and remodeled over large distances from cementum to alveolar bone. Recently, novel instruments and 3D-imaging methods have been developed that have been applied to the investigation of hard tissues including the PDL. Work from our laboratory has revealed the three-dimensional (3D) ultrastructure of PDLFs and PDL collagen bundles by focused ion beam/scanning electron microscope tomography. We have shown that PDLFs have a flat shape with long processes or a wing-like shape, while PDL bundles are a multiple-branched structure wrapped in thin sheets of PDLF cytoplasm. Furthermore, PDLFs form an extensive cellular network between the cementum and alveolar bone. The PDL cellular network is presumed to synchronize PDL fiber bundles and regulate arrays of PDL fiber bundles via gap junctions. In this review, we summarize and discuss our current 3D-histomorphometric studies of the PDL at the mesoscale level.

Extracellular Matrix Induces Periodontal Ligament Reconstruction In Vivo.

One of the problems in dental implant treatment is the lack of periodontal ligament (PDL), which supports teeth, prevents infection, and transduces sensations such as chewiness. The objective of the present study was to develop a decellularized PDL for supporting an artificial tooth. To this end, we prepared mouse decellularized mandible bone with a PDL matrix by high hydrostatic pressure and DNase and detergent treatments and evaluated its reconstruction in vivo. After tooth extraction, the decellularized mandible bone with PDL matrix was implanted under the subrenal capsule in rat and observed that host cells migrated into the matrix and oriented along the PDL collagen fibers. The extracted decellularized tooth and de- and re-calcified teeth, which was used as an artificial tooth model, were re-inserted into the decellularized mandible bone and implanted under the subrenal capsule in rat. The reconstructed PDL matrix for the extracted decellularized tooth resembled the decellularized mandible bone without tooth extraction. This demonstrates that decellularized PDL matrix can reconstruct PDL tissue by controlling host cell migration, which could serve as a novel periodontal treatment approach.

Study of Hyperbaric Oxygen Theraphy and Gold sea Cucumber (Stichopus Hermanii) 3% for Collagen Density of Rat’s with Diabetes Mellitus Induced by Porphyromonas Gingivalis Bactery

<p><strong><em>Background:</em></strong><em> Periodontitis is complication of DM in chronic state. Periodontitis susceptibility increase threefold in patient DM compared non-DM. Debridement with SRP alone is less effective in DM</em><em>. Hyperbaric Oxygen Therapy (HBO) has the effect of increasing insulin sensitivity and synthesis of collagen. Collagen is the largest extracellular matrix supporting periodontal tissue. Stchopus hermanii contains condrotin sulfate serves to inhibit the formation of mixed periodontopatogen bacteri. <strong>Purpose:</strong> To determine the therapeutic effects of HBO 2,4 ATA 3x3’ interval 5’ 7 days and stichopus hermanii against periodontal ligament collagen density in the healing process of periodontitis with DM. <strong>Materials and Methods:</strong></em><em>The experiment design is factorial design. Rats are inducted with STZ 65 mg/kgBB to make them suffer DM and are inducted with P.gingivalis </em><em>1x10<sup>9</sup>sel/ml for 5 days straight to make them suffer periodontitis</em><em>,</em><em> 20 male Wistar rats were divided into 4 groups. K0 (control group), K1(STZ+P. gingivalis), K2(STZ+P. gingivalis+Stichopus hermanii), K3(STZ+P. gingivalis+HBO), and K4(STZ+P.gingivalis+stichopus hermanii HBO) after treatment on day 52, all groups were sacrificed and done making preparations for the HPA of the mandible bone. <strong>Results:</strong> Data were analyzed using chi-square, Kruskal wallis, and Mann Whitney U with 95% significance level (p = 0.05). increase level of collagen significantly</em> K1<em>(1 ± 0.00)</em> - K2<em>(2±0.00)</em>, dan K1<em>(1 ± 0.00)</em> - K4<em>(2.5 ± 0.289)</em><em>.<strong>Conclution</strong></em><strong><em>:</em></strong><em>Therapy HBO combination of 2.4 ATA 3 x 30 </em><em>with 5 minutes air breaks for 7 days</em><em> and gel Stichopus hermanii3% effective on the Densityof collagen in the periodontal ligament healing process periodontitis with DM. <br /> <br /> </em></p><p><strong><em>Keywords:</em></strong><em>OHB, Stichopus hermanii, periodontitis with DM, collagen</em></p><p><strong><em> </em></strong></p><strong><em>Correspondence: </em></strong><em>Dian Mulawarmanti, Department of Oral Biology, Faculty of Dentistry, Hang Tuah University,Arif Rahman Hakim 150, Surabaya,Phone 031-5945864, Email: </em><a href="mailto:dian.mulawarmanti@hangtuah.ac.id"><em>dian.mulawarmanti@hangtuah.ac.id</em></a>

Three-dimensional ultrastructural analysis and histomorphometry of collagen bundles in the periodontal ligament using focused ion beam/scanning electron microscope tomography.

The periodontal ligament (PDL) is an essential tissue for tooth function. However, the 3-dimensional ultrastructure of these PDL collagen bundles on a mesoscale is not clear. We investigated the 3-dimensional ultrastructure of these collagen bundles and quantitatively analyzed their histomorphometry using focused ion beam/scanning electron microscope (FIB/SEM) tomography. The PDLs of the first mandibular molar of male C57BL/6 mice were analyzed using FIB/SEM tomography. The serial images of the collagen bundles so obtained were reconstructed. The collagen bundles were analyzed quantitatively using 3-dimensional histomorphometry. Collagen bundles of the PDL demonstrated multiple branched structures, rather than a single rope-like structure, and were wrapped in cytoplasm sheets. The structure of the horizontal fiber of the collagen bundle was an extensive meshwork. In contrast, the oblique and apical fibers of the collagen bundle showed a chain-like structure. The area and the minor and major axis lengths of cross-sections of the horizontal fiber, as determined from 3-dimensional images, were significantly different from those of the oblique and apical fibers. These findings indicate that collagen bundles in horizontal fiber areas have high strength and that the tooth is firmly anchored to the alveolar bone by the horizontal fibers, but is not secured evenly to the alveolar bone. The tooth is firmly anchored around the cervical area, creating a "slingshot-like structure." This study has provided further insights into the structure of the PDL and forms the basis for the development of more effective therapies for periodontal tissue regeneration.

Distinct post-translational features of type I collagen are conserved in mouse and human periodontal ligament.

Specifics of the biochemical pathways that modulate collagen cross-links in the periodontal ligament (PDL) are not fully defined. Better knowledge of the collagen post-translational modifications that give PDL its distinct tissue properties is needed to understand the pathogenic mechanisms of human PDL destruction in periodontal disease. In this study, the post-translational phenotypes of human and mouse PDL type I collagen were surveyed using mass spectrometry. PDL is a highly specialized connective tissue that joins tooth cementum to alveolar bone. The main function of the PDL is to support the tooth within the alveolar bone while under occlusal load after tooth eruption. Almost half of the adult population in the USA has periodontal disease resulting from inflammatory destruction of the PDL, leading to tooth loss. Interestingly, PDL is unique from other ligamentous connective tissues as it has a high rate of turnover. Rapid turnover is believed to be an important characteristic for this specialized ligament to function within the oral-microbial environment. Like other ligaments, PDL is composed predominantly of type I collagen. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including hydroxylation, glycosylation and cross-linking. The chemistry, placement and quantity of intermolecular cross-links are believed to be important regulators of tissue-specific structural and mechanical properties of collagens. Type I collagen was isolated from several mouse and human tissues, including PDL, and analyzed by mass spectrometry for post-translational variances. The collagen telopeptide cross-linking lysines of PDL were found to be partially hydroxylated in human and mouse, as well as in other types of ligament. However, the degree of hydroxylation and glycosylation at the helical Lys87 cross-linking residue varied across species and between ligaments. These data suggest that different types of ligament collagen, notably PDL, appear to have evolved distinctive lysine/hydroxylysine cross-linking variations. Another distinguishing feature of PDL collagen is that, unlike other ligaments, it lacks any of the known prolyl 3-hydroxylase 2-catalyzed 3-hydroxyproline site modifications that characterize tendon and ligament collagens. This gives PDL a novel modification profile, with hybrid features of both ligament and skin collagens. This distinctive post-translational phenotype may be relevant for understanding why some individuals are at risk of rapid PDL destruction in periodontal disease and warrants further investigation. In addition, developing a murine model for studying PDL collagen may be useful for exploring potential clinical strategies for promoting PDL regeneration.

SPARC and the N-propeptide of collagen I influence fibroblast proliferation and collagen assembly in the periodontal ligament.

The periodontal ligament (PDL) is a fibrous connective tissue that anchors tooth cementum into alveolar bone. Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein known to influence collagen fiber assembly in the PDL. In contrast, functional properties of the N-propeptide of collagen I, encoded in exon 2 of the COL1A1 gene, are poorly understood. In this study, the PDL of collagen I exon 2-deleted (wt/ko), SPARC-null (ko/wt), and double transgenic (ko/ko) mice were evaluated in terms of cellularity, collagen area, fiber morphology, and extraction force and compared to WT (wt/wt) mice. Picro sirius red staining indicated a decrease in total PDL collagen content in each of the transgenic mice compared to WT at 1 and 3 month age points. At 12 months, only SPARC-null (ko/wt) and double-null PDL demonstrated less total collagen versus WT. Likewise, an increase in thin PDL collagen fibers was observed at 1 and 3 months in each transgenic, with increases only in SPARC-null and double-null mice at 12 months. The force required for tooth extraction was significantly reduced in SPARC-null versus exon 2-deleted and WT mice, whereas double-null mice demonstrated further decreases in force required for tooth extraction. The number of proliferating fibroblasts and number and size of epithelial rests of Malassez were increased in each transgenic versus WT with double-null PDL exhibiting highest levels of proliferation and rests of Malassez at 1 month of age. Consistent with increases in PDL collagen in exon-2 deleted mice, with age, numbers of rests decreased at 12 months in this genotype. These results demonstrate for the first time a functional role of the N-propeptide in regulating collagen fiber assembly and cell behavior and suggest that SPARC and the N-propeptide of collagen I have distinct activities in regulating collagen fiber assembly and fibroblast function.

Residents' journal review

Residents' journal review

PDL Progenitor–Mediated PDL Recovery Contributes to Orthodontic Relapse

Periodontal ligament (PDL) is subjected to mechanical force during physiologic activities. PDL stem/progenitor cells are the main mesenchymal stem cells in PDL. However, how PDL progenitors participate in PDL homeostasis upon and after mechanical force is largely unknown. In this study, force-triggered orthodontic tooth movement and the following relapse were used as models to demonstrate the response of PDL progenitors and their role in PDL remodeling upon and after mechanical force. Upon orthodontic force, PDL collagen on the compression side significantly degraded, showing a broken and disorganized pattern. After force withdrawal, the degraded PDL collagen recovered during the early stage of relapse. Correspondingly, increased CD90+ PDL progenitors with suppressed expression of type I collagen (Col-I) were observed upon orthodontic force, whereas these cells accumulated at the degradation regions and regained Col-I expression after force withdrawal during early relapse. Our results further showed that compressive force altered cell morphology and repressed collagen expression in cultured PDL progenitors, which both recovered after force withdrawal. Force withdrawal–induced recovery of collagen expression in cultured PDL progenitors could be regulated by transforming growth factor–β (TGF-β), a key molecule for tissue homeostasis and extracellular matrix remodeling. More interesting, inhibiting the regained Col-I expression in CD90+ PDL progenitors by blocking TGF-β interrupted PDL collagen recovery and partially inhibited the early relapse. These data suggest that PDL progenitors can respond to mechanical force and may process intrinsic stability to recover to original status after force withdrawal. PDL progenitors with intrinsic stability are required for PDL recovery and consequently contribute to early orthodontic relapse, which can be regulated by TGF-β signaling.

Altered distribution of HMGB1 in the periodontal ligament of periostin-deficient mice subjected to Waldo's orthodontic tooth movement.

Periostin is essential for the integrity and function of the periodontal ligament (PDL), and periostin knockout is related to an enhanced inflammatory status in PDL. High mobility group box 1 (HMGB1), a late inflammatory cytokine, is up-regulated in PDL cells in response to mechanical stress. This study aimed to investigate the effect of periostin deficiency (Pn-/-) on HMGB1 expression in PDL during orthodontic tooth movement. We used 8-week-old male mice homozygous for the disrupted periostin gene and their wild-type (WT) littermates. Tooth movement was performed according to Waldo's method, in which 0.5-mm-thick elastic bands were inserted between the first and second upper molars of anesthetized mice. After 3 days of mechanical loading, mice were fixed by transcardial perfusion of 4% paraformaldehyde in phosphate buffer, and the maxilla was extracted for histochemical analyses. Compared with the WT group, Pn-/- mice showed higher basal expression of HMGB1 in the absence of mechanical loading. Following 3 days of orthodontic tooth movement, the PDL in the compression side of both groups was almost replaced by cell-free hyaline zones, and Pn-/- mice showed a much wider residual PDL than WT mice. In the tension side, the number of HMGB1-positive cells in PDL in both Pn-/- and WT groups increased remarkably without a significant difference between the two groups. Our findings suggest an inhibitory effect of periostin on HMGB1 production by PDL and confirmed the critical role of periostin in integrity of PDL collagen fibrils during orthodontic tooth movement, although mechanical loading is the predominant stimulant of HMGB1 expression relative to periostin deficiency.

Decreased Mechanical Strength and Collagen Content in SPARC-Null Periodontal Ligament Is Reversed by Inhibition of Transglutaminase Activity.

The periodontal ligament (PDL) is a critical tissue that provides a physical link between the mineralized outer layer of the tooth and the alveolar bone. The PDL is composed primarily of nonmineralized fibrillar collagens. Expression of secreted protein acidic and rich in cysteine (SPARC/osteonectin), a collagen-binding matricellular protein, has been shown to be essential for collagen homeostasis in PDL. In the absence of SPARC, PDL collagen fibers are smaller and less dense than fibers that constitute WT PDL. The aim of this study was to identify cellular mechanisms by which SPARC affected collagen fiber assembly and morphology in PDL. Cross-linking of fibrillar collagens is one parameter that is known to affect insoluble collagen incorporation and fiber morphology. Herein, the reduction in collagen fiber size and quantity in the absence of SPARC expression was shown to result in a PDL with reduced molar extraction force in comparison to that of WT mice (C57Bl/6J). Furthermore, an increase in transglutaminase activity was found in SPARC-null PDL by biochemical analyses that was supported by immunohistochemical results. Specifically, collagen I was identified as a substrate for transglutaminase in PDL and transglutaminase activity on collagen I was found to be greater in SPARC-null tissues in comparison to WT. Strikingly, inhibition of transglutaminase activity in SPARC-null PDL resulted in increases in both collagen fiber thickness and in collagen content, whereas transglutaminase inhibitors injected into WT mice resulted in increases in collagen fiber thickness only. Furthermore, PDL treated with transglutaminase inhibitors exhibited increases in molar extraction force in WT and in SPARC-null mice. Thus, SPARC is proposed to act as a critical regulator of transglutaminase activity on collagen I with implications for mechanical strength of tissues.