Non-mineralized Tissue Research Articles

Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering

In developmental biology, gradients of bioactive signals direct the formation of structural transitions in tissue that are key to physiological function. Failure to reproduce these native features in an in vitro setting can severely limit the success of bioengineered tissue constructs. In this report, we introduce a facile and rapid platform that uses magnetic field alignment of glycosylated superparamagnetic iron oxide nanoparticles, pre-loaded with growth factors, to pattern biochemical gradients into a range of biomaterial systems. Gradients of bone morphogenetic protein 2 in agarose hydrogels were used to spatially direct the osteogenesis of human mesenchymal stem cells and generate robust osteochondral tissue constructs exhibiting a clear mineral transition from bone to cartilage. Interestingly, the smooth gradients in growth factor concentration gave rise to biologically-relevant, emergent structural features, including a tidemark transition demarcating mineralized and non-mineralized tissue and an osteochondral interface rich in hypertrophic chondrocytes. This platform technology offers great versatility and provides an exciting new opportunity for overcoming a range of interfacial tissue engineering challenges.

Life-Threatening Hypocalcemia in A Patient with highly suspected osteomalacia: A Case Report

ABSTRACT Objective: Osteomalacia is a metabolic bone disease characterized by impaired mineralization with increased non-mineralized osteoid tissue, increased frailty, and reduced bone mineral density. A common cause of osteomalacia in adults and the elderly is severe deficiency of vitamin D, which leads to chronic hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism. The objective of this case report is to describe an unusual clinical presentation of osteomalacia, consisting of life-threatening acute hypocalcemia. Methods: Clinical, laboratory, and imaging data are presented. Results: We report the case of a 65-year-old man that showed symptoms and signs of severe and prolonged hypocalcemia due to unrecognized vitamin D deficiency. He presented at the emergency room reporting abdominal pain and vomiting since the evening before. Blood tests showed increased levels of rhabdomyolysis markers, severe hypocalcemia, hypophosphatemia, hypomagnesemia, normal renal function, elevated levels of alkal...

Effect of total sonicated Aggregatibacter actinomycetemcomitans fragments on gingival stem/progenitor cells.

BackgroundAggregatibacter-actinomycetemcomitans (A.actinomycetemcomitans) are strongly associated with localized-aggressive-periodontitis (LAgP). The study’s aim was to test for the first time the effect of total sonicated A.actinomycetemcomitans-bacterial-fragments on gingival mesenchymal stem/progenitor cells’ (G-MSCs) proliferation and regenerative gene expression in-vitro.Material and MethodsG-MSCs were isolated, characterized, expanded and stimulated by total sonicated A.actinomycetemcomitans-bacterial-fragments (0 (negative-control), 15, 60, 120 and 240µg/ml; serovar-b; n=6/group). Cellular proliferation and NF-κβ (NFKB1), Alkaline Phosphatase (ALPL), Collagen-I (COL1A1), Collagen-III (COL3A1), Osteonectin (SPARC) and Osteopontin (SPP1) m-RNA expression were assessed via reverse-transcription-polymerase-chain-reaction (RT-PCR) at 24, 48 and 72 hours and CFUs-ability evaluated at twelve days.ResultsG-MSCs demonstrated stem/progenitor cells’ characteristics. A.actinomycetemcomitans-bacterial-fragments (up to 72 hours) resulted in marked G-MSCs’ proliferation over-time (p<0.001) and elevated NFKB1 (p=0.017), COL1A1 (p=0.025), SPARC (p=0.025), decreased ALPL (p=0.017), with no significant differences for COL3A1 and SPP1 expression or stimulation times (p>0.05; Friedman-test). Longer-term stimulation for twelve days reduced G-MSCs’ CFUs.ConclusionsSonicated A.actinomycetemcomitans-bacterial-fragments’ exert beneficial short-term effects on G-MSCs’ proliferative and non-mineralized tissue forming aptitude. Results shed new light on the importance of periodontal treatment for LAgP patients, using power driven sonic/ultrasonic devices, which, in addition to reducing the subgingival microbial load, produces cell-stimulatory A.actinomycetemcomitans-bacterial-fragments, with positive attributes on tissue reparative/regenerative responses of tissue resident stem/progenitor cells in their niche. Key words:Ultrasonic, Aggregatibacter actinomycetemcomitans, stem cells, gingiva, Aggressive periodontitis.

Bone Healing in Extraction Sockets Covered With Collagen Membrane Alone or Associated With Porcine-Derived Bone Graft: a Comparative Histological and Histomorphometric Analysis.

ABSTRACTObjectivesThe present paper reports data of a randomized study aimed to analyse and compare the histologic and histomorphometric aspects of bone healing in extraction sites covered with collagen membrane alone or associated with porcine-derived bone graft.Material and MethodsThirty patients, with single extraction sockets without severe bone wall defects in the premolar/molar region, were included. Ten extraction sockets were grafted with porcine-derived bone and covered with collagen membrane (group 1), 10 sites were covered with collagen membrane alone (group 2), and 10 sites healed spontaneously (group 3). After 4 months of healing, 26 (8 in group 1, 9 in group 2, and 9 in group 3) bone core specimens were harvested for histologic evaluation, then dental implants were placed.ResultsSites in the group 1 and in the group 2 showed similar histologic and histomorphometric results without significantly differences in the percentage of vital bone (57.43% [SD 4.8] vs. 60.01% [SD 3.2]), and non-mineralized connective tissue 22.99% (SD 5.3) vs. 18.53% (SD 6.2). In group 1 a 16.57% (SD 3.8) of residual material was found. ConclusionsResults showed that the use of collagen membrane alone or associated to porcine-derived bone improves the healing bone process compared to that of extraction sites spontaneously healed. Moreover, histomorphometric data related to bone quality, indicated that extraction sites without severe walls defects and with a vestibular bone thickness > 1.5 mm, treated with a low resorbtion rate collagen membrane alone, do not need more than 4 months for dental implant insertion.

Use of Bone Allograft With or Without Bone Marrow Aspirate Concentrate in Appositional Reconstructions: A Tomographic and Histomorphometric Study.

To evaluate bone allograft associated to bone marrow aspirate concentrate (BMAC), in maxillary reconstructions. Ten patients with alveolar bone deficiency in the anterior maxilla were randomly divided into control group (CG) and test group (TG). A bone block allograft was placed in both groups, but the graft was impregnated with BMAC only in TG. Computed tomography was performed 7 days (T1) and 6 months (T2) after the grafting procedure, and volume and bone density measurements were carried out. Histomorphometric analysis was performed at T2. Bone volume loss from T1 to T2 was significant only in TG. The bone density in the buccal region of the graft was significantly higher in TG than in CG. There was no significant difference between the groups, in respect to mineralized tissue (MT) and nonmineralized tissue (NMT), in that MT values were 37.77% ± 15.19% and 43.85% ± 10.94%, and NMT values were 62.15% ± 14.90% and 56.30% ± 10.72%, respectively, for CG and TG. The intragroup difference for the MT/NMT ratio was statistically significant in CG but not significant in TG. The use of BMAC resulted in an improved pattern of bone formation, with higher bone density in the peripheral regions of the graft.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and µCT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

Histomorphometric analyses of area fraction of different ratios of Bio-Oss® and bone prior to grafting procedures - An in vitro study to demonstrate a baseline.

The objective of this study was to estimate the area fraction of different ratios of Bio-Oss® and bone, prior to grafting in an in vitro model to demonstrate a histomorphometric baseline. Bio-Oss® particles were mixed with autogenous bone from pig jaw in three different ratios (50:50, 80:20 and 100:0) and packed in rice paper in a standardized procedure. Histomorphometric analyses were performed in 25 specimens and 74 regions of interest. The area percentage of Bio-Oss® , bone, and non-mineralized tissue (NMT) were calculated. Results were reported as mean values and 95% confidence interval (CI). The mean area fraction of Bio-Oss® was 20.6% (CI: 18.2-23) in the 50:50 mixture, 33.6% (CI: 29.7-37.6) in the 80:20 mixture, and 43.4% (CI: 40.5-46.3) in the 100:0 mixture. The mean area fraction of NMT was 60.5% (CI: 57.9-63.1) in the 50:50 mixture, 59.6% (CI: 56.4-62.7) in the 80:20 mixture, and 56.6% (CI: 53.7-59.5) in the 100:0 mixture. The mean area fraction of bone was 18.9% (CI: 16.9-20.9) in the 50:50 mixture and 6.8% (CI: 5-8.6) in the 80:20 mixture. There is a great difference in the clinically estimated percentage and the histomorphometrically evaluated percentage of Bio-Oss® at baseline, prior to grafting. The area fraction of different tissues presented in this study may be beneficial as guidance for histomorphometrical baseline calculations when different mixtures of Bio-Oss® and autogenous bone are used as grafting materials.

Histopathological comparison of healing after maxillary sinus augmentation using xenograft mixed with autogenous bone versus allograft mixed with autogenous bone

To compare the clinical and histologic outcomes of two different grafting materials (allograft and xenograft) when combined with autogenous bone and covered with a collagen membrane for sinus augmentation. A parallel case series of fourteen patients in need of a unilateral sinus augmentation was evaluated in this study. Seven patients received a graft composed by autologous cortical bone (ACB) and anorganic bovine bone in a ratio of 1:1; the other seven patients received ACB mixed with an allograft in the same ratio. Bone biopsies were obtained 6months after sinus augmentation at the time of implant placement. Comparative histomorphometrical, histopathological, and immunohistochemical analyses were conducted and statistically analyzed. After 12months of functional loading, all implants in both groups were clinical and radiographically successful. Histomorphometrically, although the initial bone formation was not significantly different between groups (new mineralized tissue: 41.03(12.87)% vs. 34.50(13.18)%, p=.620; allograft vs. xenograft groups), the graft resorbed faster in the allograft group (remnant graft particles: 9.83[7.77]% vs. 21.71[17.88]%; p=.026; allograft vs. xenograft groups). Non-mineralized tissue did not statistically differ either (49.00[14.32]% vs. 43.79[19.90]%; p=.710; allograft vs. xenograft groups). The histologic analyses revealed higher cellular content, four times more osteoid lines, and higher vascularization in the xenograft group. Musashi-1 (mesenchymal stromal cell marker) was also more intensively expressed in the xenograft group (p=.019). Both composite grafts generate adequate substratum to receive dental implants after healing. Compared with the xenograft composite, allograft composite shows faster turnover and a quicker decrease in biological action after 6months.

Small Integrin-Binding Ligand N-Linked Glycoproteins (Siblings): A Study On Human Salivary Gland Cancer

Salivary gland carcinomas constitute a rare but deadly group of head and neck cancers, but timely diagnosis is often delayed due to inherent variability in etiology, heterogeneity and histopathological characterization. SIBLINGs are a family of secreted glycophosphoproteins that include osteopontin (OPN), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and matrix extracellular phosphoglycoprotein (MEPE). SIBLINGs were first discovered in bone and teeth, and were considered to be exclusively expressed in mineralized tissue. In addition to mineralized tissue, SIBLINGs have now been shown to have variable expression in normal, non-mineralized tissue and in cancers. However, there have been no studies evaluating SIBLING expression in human salivary gland cancers. Our study tested the hypothesis that SIBLINGs, specifically, BSP, DSPP and OPN, would be significantly overexpressed in human salivary gland cancer. We also hypothesized that the cancer secretome would influence SIBLING expression in normal salivary gland cells. Methods: Normal and cancerous human salivary gland tissue obtained from the NDRI were processed using routine immunohistochemistry techniques to evaluate expression of BSP, DSP, and OPN. In addition normal HSG cell line and cancer HTB-41 cell line were evaluated using immunofluorescence techniques to localize expression of BSP, DSP and OPN. Normal HSG, cancer HTB-41 and HSG* cells (normal HSG cells exposed to a cancer HTB-41 secretome) were propagated using routine cell culture techniques for 24, 48, and 72 hours. Western blotting techniques were utilized ii to quantify and compare SIBLING protein expression levels in HSG, HTB-41 and HSG* cells. Normal HSG, cancer HTB-41, and HSG* cells were processed via immunoflourescence in order to observe localization of SIBLINGs. Results: Immunohistochemistry and western blot showed increased expression of SIBLINGs in human salivary gland cancers. Furthermore, immunoflourescence revealed distinct localization of SIBLING proteins in HSG and HTB-41 cell lines. In terms of HSG*, it was found that cells exposed to cancer secretome exhibited similar SIBLING expression to HTB-41. Conclusion: Our studies confirm that SIBLING proteins are selectively expressed in human salivary gland cancer. Also, the cancer secretome is found to affect SIBLING expression in normal cells, similar to HTB-41 cancer cell lines.

Guided bone regeneration at zirconia and titanium dental implants: a pilot histological investigation.

To test whether guided bone regeneration (GBR) of peri-implant defects at zirconia (ZrO2 ) implants differs from GBR at titanium (Ti) implants regarding the bone integration of the implant and of the grafting material. Maxillary premolars and molars were extracted in seven dogs. After 5months, four semi-saddle bone defects were created in each maxilla. Implant placement and simultaneous GBR were performed using the following randomly assigned modalities: (1) ZrO2 implant+deproteinized bovine bone mineral (DBBM) granules+a collagen membrane (CM), (2) ZrO2 implant+DBBM with 10% collagen matrix+CM, (3) ZrO2 implant+DBBM block+CM, and (4) Ti implant+DBBM granules+CM. After 3months, one central histological section of each site was prepared. Histomorphometrical assessments were performed evaluating the augmented area (AA) within the former bone defect (primary outcome), the area of new bone (NB), bone substitute (BS), and non-mineralized tissue (NMT) within AA in mm2 . In addition, the distance between the most coronal bone-to-implant contact and the margin of the former bone defect (fBIC-DEF), and the bone-to-implant contact fraction (BIC) were measured in mm. AA measured 8.6±4.0mm2 for ZrO2 implant+DBBM granules, 4.7±1.6mm2 for ZrO2 implant+DBBM-collagen, 5.1±1.9mm2 for ZrO2 implant+DBBM block, and 7.6±2.8mm2 for Ti implant+DBBM granules. There were no statistically significant differences between the treatment modalities (P>0.05). NB reached 2.0±1.7mm2 for ZrO2 implant+DBBM granules, 0.9±0.9mm2 for ZrO2 implant+DBBM-collagen, 2.1±0.9mm2 for ZrO2 implant+DBBM block, and 0.8±0.6mm2 for Ti implant+DBBM granules. fBIC-DEF amounted to 2.1±1.7mm2 for ZrO2 implant+DBBM granules, to 2.7±1.1mm2 for ZrO2 implant+DBBM-collagen, to 2.9±1.2mm2 for ZrO2 implant+DBBM block, and to 3.4±0.4mm2 for Ti implant+DBBM granules. BIC measured 70±19% for ZrO2 implant+DBBM granules, 69±22% for ZrO2 implant+DBBM-collagen, 77±30% for ZrO2 implant+DBBM block, and 66±27% for Ti implant+DBBM granules. The findings of the present pilot study suggest that zirconia and titanium implants grafted with DBBM granules and covered with a collagen membrane do not perform differently regarding the augmented ridge contour, the NB formation, and the implant osseointegration.

Histomorphometric and immunohistochemical evaluation of collagen containing xenogeneic bone blocks used for lateral bone augmentation in staged implant placement

BackgroundThe osteoconductive properties of collagen containing xenogeneic bone blocks (CCXBB) remain unclear. The aim of this prospective single-arm clinical study was to assess the histological outcomes of CCXBB blocks used as bone replacement grafts for lateral bone augmentation procedures.MethodsIn 15 patients with severe horizontal alveolar ridge resorption, lateral augmentation procedures were performed using CCXBB as bone replacement grafts. Twenty-six weeks postoperatively, a re-entry procedure was performed to evaluate the bone width for adequate implant placement and two histological specimens were retrieved from each patient, one being processed for ground sectioning and the other for decalcified paraffin-included sections. In non-decalcified sections, the relative proportions occupied by bone, biomaterials, and connective tissue present in the biopsies were identified. In de-calcified sections, structures and cells positive for osteopontin (OPN), tartrate-resistant acid phosphatase activity (TRAP), osteocalcin (OSC), and alkaline phosphatase (ALP) were assessed.ResultsSoft tissue dehiscence occurred during the follow-up in 5 out of 15 patients (33.3%). The mean crest width at baseline was 2.78 mm (SD 0.57) and the mean crest width at re-entry was 6.90 mm (SD 1.22), with a mean ridge width increase of 4.12 mm (SD 1.32). Twenty-six bone biopsies were obtained from 13 patients. Histomorphometric analysis showed a mean of 26.90% (SD 12.21) of mineralized vital bone (MVB), 21.37% (SD 7.36) of residual CCXBB, 47.13% (SD 19.15) of non-mineralized tissue, and 0.92% of DBBM. The immunohistochemical analysis revealed a large number of OPN-positive cells 8.12% (SD 4.73), a lower proportion of TRAP positive multinuclear cells 5.09% (SD 4.91), OSC-positive cells 4.09% (SD 4.34), and a limited amount of ALP positive cells 1.63% (SD 2).ConclusionsCCXBB achieved significant horizontal crestal width allowing for staged implant placement in most of the patients. In light of the histological outcomes and implant failures, special attention must be placed to prevent soft tissue dehiscence when CCXBB is used in severe atrophic alveolar crests.

A prospective randomized controlled trial of two‐window versus solo‐window technique by lateral sinus floor elevation in atrophic posterior maxilla: Results from a 1‐year observational phase

Implant failures are more common when multiple missing posterior teeth need lateral sinus floor elevation owing to inadequate tissue maturation after grafting. Effects of lateral window dimensions on vital bone formation have rarely been compared. To compare endo-sinus bone formation between two- and solo-window techniques to rehabilitate multiple missing posterior teeth that need substantial augmentation. Patients with severely atrophic posterior maxilla were randomized to receive lateral sinus floor elevation via solo or two bony windows. Bone core specimens harvested from lateral aspect of the augmentation sites were histomorphometrically analyzed. Proportions of mineralized bone (MB), bone substitute materials (BS), and nonmineralized tissue (NMT) were quantified. Twenty-one patients underwent 23 maxillary sinus augmentations. One patient in each group dropped out during the follow-up period. Lateral window dimensions were 81.65 ± 4.59 and 118.04 ± 19.53 mm2 in the test and control groups, respectively. Histomorphometric analysis revealed mean MB of 42.32% ± 13.07% and 26.00% ± 15.23%, BS of 40.34% ± 9.52% and 60.03% ± 10.13%, and NMT of 18.14% ± 14.24% and 14.75% ± 10.38% in test and control groups, respectively, with significant differences. The two-window technique could facilitate faster maturation and consolidation of the grafted volume and is an effective alternative for rehabilitation of severely atrophic posterior maxilla with multiple missing posterior teeth.

Primary ridge augmentation with collagenated xenogenic block bone substitute in combination with collagen membrane and rhBMP-2: a pilot histological investigation.

The aim of this pilot study was to test whether a porcine collagenated bone substitute block (PCBB) and collagen membrane (CM) loaded with bone morphogenetic protein-2 (BMP-2) used for horizontal ridge augmentation differ from PCBB and CM without BMP-2 regarding the osseointegration of the grafting material and the maintenance of the ridge contour. Two semi-saddle bone defects were created in each side of the mandible of six dogs. The defects were randomly allocated to receive one of the following treatments: bone augmentation using (1) PCBB, (2) PCBB loaded with BMP-2 (PCBB-BMP2), (3) PCBB+CM and (4) PCBB+CM loaded with BMP-2 (PCBB+CM-BMP2). After 12weeks, one titanium implant was inserted into every site. After 8weeks, one central histological section of each site was prepared. Histomorphometrical assessments were performed evaluating the augmented area (AA), the area of new bone (NB) (primary outcome), residual bone substitute (BS) and non-mineralized tissue (NMT) within AA in mm2 . In addition, the most coronal and the most buccal localizations of new bone and residual bone substitute, and the most coronal bone-to-implant contact were measured in mm. Clinically, all PCBB were firmly integrated and permitted implant placement. All the implants osseointegrated and exhibited complete hard-tissue coverage of the buccal surface. Bone ingrowth always reached the central portions of PCBB. AA measured 10.4±4.2mm2 for PCBB, 11.8±2.8mm2 for PCBB-BMP2, 9.8±2.9mm2 for PCBB+CM and 8.5±2.2mm2 for PCBB+CM-BMP2. Only the difference between PCBB-BMP2 and PCBB+CM-BMP2 was statistically significant (P=0.031). NB reached 2.3±1.3mm2 for PCBB, 2±0.5mm2 for PCBB-BMP2, 2.7±1.2mm2 for PCBB+CM and 1.8±0.7mm2 for PCBB+CM-BMP2. There were no statistically significant differences regarding NB, the most coronal and the most buccal localizations of new bone, residual bone substitute and bone-to-implant contact (P>0.05). The addition of BMP-2 to PCBB or CM used for horizontal ridge augmentation did not render a statistically significant improvement in the maintenance of the augmented ridge contour and the new bone formation. PCBB with and without CM showed pronounced bone ingrowth and capacity to maintain the augmented ridge contour. In all the regions previously augmented with PCBB, the implants successfully integrated and presented with complete hard-tissue coverage.

Cationic osteogenic peptide P15-CSP coatings promote 3-D osteogenesis in poly(epsilon-caprolactone) scaffolds of distinct pore size.

P15-CSP is a biomimetic cationic fusion peptide that stimulates osteogenesis and inhibits bacterial biofilm formation when coated on 2-D surfaces. This study tested the hypothesis that P15-CSP coatings enhance 3-D osteogenesis in a porous but otherwise hydrophobic poly-(ɛ-caprolactone) (PCL) scaffold. Scaffolds of 84 µm and 141 µm average pore size were coated or not with Layer-by-Layer polyelectrolytes followed by P15-CSP, seeded with adult primary human mesenchymal stem cells (MSCs), and cultured 10 days in proliferation medium, then 21 days in osteogenic medium. Atomic analyses showed that P15-CSP was successfully captured by LbL. After 2 days of culture, MSCs adhered and spread more on P15-CSP coated pores than PCL-only. At day 10, all constructs contained nonmineralized tissue. At day 31, all constructs became enveloped in a "skin" of tissue that, like 2-D cultures, underwent sporadic mineralization in areas of high cell density that extended into some 141 µm edge pores. By quantitative histomorphometry, 2.5-fold more tissue and biomineral accumulated in edge pores versus inner pores. P15-CSP specifically promoted tissue-scaffold integration, fourfold higher overall biomineralization, and more mineral deposits in the outer 84 µm and inner 141 µm pores than PCL-only (p < 0.05). 3-D Micro-CT revealed asymmetric mineral deposition consistent with histological calcium staining. This study provides proof-of-concept that P15-CSP coatings are osteoconductive in PCL pore surfaces with 3-D topography. Biomineralization deeper than 150 µm from the scaffold edge was optimally attained with the larger 141 µm peptide-coated pores. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2171-2181, 2017.

Guided bone regeneration of non-contained mandibular buccal bone defects using deproteinized bovine bone mineral and a collagen membrane: an experimental invivo investigation.

The aim of this pre-clinical invivo study was to analyse different stages of wound healing after guided bone regeneration in non-contained mandibular buccal bone defects. Eighteen female beagle dogs, between 1.5 and 2years old, were used. Buccal bone defects were created in the mandible following extraction of the mesial roots of M1, P4, the distal root of P3 and booth roots of P2. Augmentation procedures of the healed defects were performed 3months later using a bone replacement graft (T1), an absorbable collagen membrane (T2) or a combination of both procedures (T3). Using a randomized block study design, four stages of healing in two groups of dogs were examined (4days, 2, 6weeks and 3months). The animals were euthanized, and biopsies obtained at the end of each of the study periods were prepared for histological examination. The different reconstructive procedures resulted in regenerated tissue compartments of varying size that contained newly formed bone, non-mineralized tissue and bone augmentation biomaterial when a bone replacement graft was used. While the proportions of mineralized tissue increased and non-mineralized tissue decreased over time in the three groups, the changes in proportions of the DBBM material were small. Initial defect depth, healing time and treatment group significantly influenced the percentage of mineralized tissue obtained. The multivariate multilevel analysis showed that significantly larger area proportions of mineralized tissue were obtained when the T2 sites were compared with T1 and T3 sites, what highlights the importance of the barrier membrane effect for attaining new bone formation. Only in the larger size defects (M1) total ROI at T3 and T1 sites was significantly larger than at T2, what highlights the importance of using a bone replacement graft as a space maintenance scaffold. It is suggested that healing following augmentation of non-contained buccal bone defects was characterized by a gradual shift in the relative proportions of non-mineralized and mineralized tissue components.

Evaluation of high-resolution In Vivo MRI for longitudinal analysis of endochondral fracture healing in mice.

Mice are extensively used for experimental bone-healing studies. However, there are few established nondestructive in vivo techniques for longitudinal fracture-healing analysis in mice, including in vivo micro-computed tomography (μCT) and radiography. Importantly, none of the established methods can discriminate between non-mineralized fibrous tissue and cartilage in the soft fracture callus. Therefore, the objective was to establish high-resolution in vivo magnetic resonance imaging (MRI) for the longitudinal assessment of soft callus formation during bone healing in mice. C57BL/6J mice received a femur osteotomy stabilized using an external fixator and were randomly assigned to five groups. Group 1 mice were scanned three times longitudinally during fracture healing using an optimized MRI scanning protocol to establish an algorithm to characterize the different fracture-callus tissues. Mice of groups 2–4 were scanned once on day 10, 14 or 21, respectively, euthanized after scanning and their femurs subjected to ex vivo μCT and histomorphometric analysis to compare the data assessed by MRI with μCT and histology. Control group 5 mice were not scanned. After 28 days, mice of groups 1 and 5 were euthanized and the fracture-healing outcome was evaluated by bending-test, μCT and histology to determine whether the repeated anesthesia, handling and the MRI measurements themselves influenced fracture healing. The callus-tissue values determined by MRI were mostly comparable to those obtained by μCT and histomorphometric analysis. However, at time points characterized by small relative bone or cartilage areas, MRI measurements were weakly comparable to histomorphometric data, possibly due to the inferior spatial resolution. Importantly, at the early and intermediate phases of healing, cartilage and fibrous-tissue values obtained by MRI were highly accurate. Furthermore, repeated anesthesia, handling and MRI scans did not impact bone healing. Therefore, we demonstrated the feasibility of high-resolution in vivo MRI for longitudinal assessment of soft callus formation during murine endochondral fracture healing.

Bone Regeneration Using Adipose-Derived Stem Cells with Fibronectin in Dehiscence-Type Defects Associated with Dental Implants: An Experimental Study in a Dog Model.

To determine the bone regeneration potential of a ceramic biomaterial coated with fibronectin and adipose-derived stem cells covered in three-wall critical-size defects associated with dental implants. In a total of 18 dogs, four dehiscence-type and critical-size defects were created surgically in the edentulous alveolar ridge with the simultaneous placement of dental implants. Defects were randomly regenerated using biomaterials coated with particulate ß-tricalcium phosphate (β-TCP), β-TCP with fibronectin (Fn) (β-TCP-Fn), and β-TCP with a combination of Fn and autologous adipose-derived stem cells (ADSCs) (β-TCP-Fn-ADSCs), leaving one defect as the control. The animals were divided into three groups according to the time of euthanasia (1, 2, or 3 months). Statistically significant differences between the three study groups (β-TCP, β-TCP-Fn, β-TCP-Fn-ADSCs) and the control group in the total area of bone regeneration and mineralized and nonmineralized tissue at 1, 2, and 3 months of healing were not observed. At 2 months, defects treated with β-TCP-Fn-ADSCs showed a significant decrease in the percentage of bone-to-implant contact (BIC) as compared with the β-TCP-Fn (P = .041) and control (P = .012) groups. At 3 months of healing, however, significant differences in BIC between the three study groups and controls were not found (P = .388). The use of ADSCs in the bone regeneration processes of dehiscencetype defects associated with simultaneous implant insertion does not seem to improve the area of bone regeneration or the percentage of BIC compared with other biomaterials or the control alveolar defect.

Histomorphometric Results After Postextraction Socket Healing with Different Biomaterials: A Systematic Review of the Literature and Meta-Analysis.

The aim of this systematic review was to investigate which material is the most effective bone substitute for alveolar bone healing by evaluating histomorphometric outcomes after healing of postextraction sockets in humans. A manual and electronic search (PubMed, EMBASE, Cochrane Library) was performed using a search string prepared ad hoc. Data were statistically analyzed by calculating weighted mean percentage of new bone formation (primary outcome) and weighted mean percentage of residual biomaterial, soft/connective tissue, and nonmineralized tissue (secondary outcomes) in the biopsies. A meta-analysis of the included randomized controlled trials (RCTs) was performed. A total of 802 papers were screened. After application of the inclusion and exclusion criteria, a total of 40 articles were included in the quantitative synthesis while 11 were included in the meta-analysis of comparative studies. The evaluation of comparative studies with empty sites as control showed that bovine bone could lead to a lower proportion of new bone formation compared to sites left to heal spontaneously (P < .00001). Magnesium-enriched hydroxyapatite and porcine bone showed a significantly higher percentage of new bone compared to control sites (P < .00001). Grafting with an allograft did not lead to a higher percentage of new bone formation in comparison with control sites (P = .09). There was no evidence for the superiority of a given biomaterial over the others in terms of new bone formation. While calcium sulphate and beta-tricalcium phosphate resorbed faster than other biomaterials, xenografts showed a lower resorption rate than allografts. Comparative studies suggested that bovine bone was related to a lower proportion of new bone volume compared to sites left to heal spontaneously, while porcine bone and magnesium-enriched hydroxyapatite were related to higher new bone volume. Allograft was not related to higher new bone volume than sites healed without any biomaterial.

Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis.

The periodontal ligament (PDL) is a non-mineralized connective tissue that exists between the alveolar bone and root surface cementum and plays important roles in tooth function. The PDL harbors a remarkable reserve of multipotent stem cells, which maintain various types of cells. However, the sources of these stem cells, other than their developmental origin, are not well understood. To elucidate the recruitment of bone marrow (BM)-derived stem cells in the PDL, green fluorescent protein (GFP)-expressing BM-derived cells were transplanted into the femoral BM of immunodeficient rats, and the distribution and expression of stem cell markers in the PDL were analyzed in vivo. To evaluate the functional significance of BM-derived cells to the PDL, tooth replantation was performed and the expression of stromal cell-derived factor (SDF)-1, a critical chemotactic signal for mesenchymal stem cell recruitment, was analyzed. To confirm the SDF-1-dependency of BM-derived cell migration to the PDL, PDL-conditioned medium (CM) was prepared, and BM-derived cell migration was analyzed using a transwell culture system. Four weeks after cell transplantation, GFP-positive cells were detected in the PDL, and some of them were also positive for stem cell markers (i.e., CD29, SSEA4, and αSMA). Seven days after tooth replantation, the number of GFP- and SDF-1-positive cells significantly increased in PDL. Concurrently, the concentration of SDF-1 and the number of colony-forming units of fibroblasts in peripheral blood were increased. BM-derived cell migration increased in PDL-CM and was inhibited by an inhibitor of C-X-C chemokine receptor type 4 (CXCR4), an SDF-1 receptor. These results indicate that stem cells and their progeny in PDL are not only derived from their developmental origin but are also supplied from the BM via the blood as the need arises. Moreover, this BM-derived cell recruitment appears to be regulated, at least partially, by the SDF-1/CXCR4 axis.

The influence of curvature on three-dimensional mineralized matrix formation under static and perfused conditions: an in vitro bioreactor model.

Bone remodelling is the continuous turnover of bone by resorption and formation. It is controlled by interstitial fluid flow sensed by osteocytes. The refilling of bone resorption sites has been shown to be curvature driven. In vitro, curvature influences tissue growth and cytoskeletal arrangements under static and perfused conditions. Nevertheless, this has only been demonstrated for non-mineralized tissue in limited three-dimensional volumes. This study aims at investigating the influence of three different channel curvatures (S, -2.00 mm-1; M, -1.33 mm-1; L, -0.67 mm-1) on mineralized tissue formation in three dimensions under static and perfused conditions. The ingrowth of mineralized tissue into the channels was dependent on curvature and was higher under perfusion (M and S channels). L channels were not closed in any group compared with partially (static) or fully (perfused) closed M and S channels. Mineralized tissue morphology was cortical-like in static samples and trabecular-like in perfused samples. Our results suggest that the three-dimensional in vitro model presented is not only able to reveal effects of curvature on mineralized tissue formation, but may be used as an in vitro model for critical size defects in trabecular or cortical bone.