Methods to Enable Spatial Transcriptomics of Bone Tissues.

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with normal development and disease pathology. Spatial transcriptomics is a powerful method that enables the analysis of the whole transcriptome within tissue samples, thus providing information about the cellular gene expression and the histological context in which the cells reside. While this method has been extensively utilized for many soft tissues, its application for the analyses of hard tissues such as bone has been challenging. The major challenge resides in the inability to preserve good quality RNA and tissue morphology while processing the hard tissue samples for sectioning. Therefore, a method is described here to process freshly obtained bone tissue samples to effectively generate spatial transcriptomics data. The method allows for the decalcification of the samples, granting successful tissue sections with preserved morphological details while avoiding RNA degradation. In addition, detailed guidelines are provided for samples that were previously paraffin-embedded, without demineralization, such as samples collected from tissue banks. Using these guidelines, high-quality spatial transcriptomics data generated from tissue bank samples of primary tumor and lung metastasis of bone osteosarcoma are shown.

ALDH1A1 Gene Expression and Cellular Copper Levels between Low and Highly Metastatic Osteosarcoma Provide a Case for Novel Repurposing with Disulfiram and Copper.

Aldehyde dehydrogenase 1A1 (ALDH) is a cancer stem cell marker highly expressed in metastatic cells. Disulfiram (Dis) is an FDA-approved antialcoholism drug that inhibits ALDH and has been studied as a candidate for drug repurposing in multiple neoplasia. Dis cytotoxicity in cancer cells has been shown to be copper-dependent, in part due to Dis's ability to function as a bivalent metal ion chelator of copper (Cu). The objectives of this research were to test ALDH expression levels and Cu concentrations in sarcoma patient tumors and human osteosarcoma (OS) cell lines with differing metastatic phenotypes. We also sought to evaluate Dis + Cu combination therapy in human OS cells. Intracellular Cu was inversely proportional to the metastatic phenotype in human OS cell lines (SaOS2 > LM2 > LM7). Nonmetastatic human sarcoma tumors demonstrated increased Cu concentrations compared with metastatic tumors. qPCR demonstrated that ALDH expression was significantly increased in highly metastatic LM2 and LM7 human OS cell lines compared with low metastatic SaOS2. Tumor cells from sarcoma patients with metastatic disease displayed significantly increased ALDH expression compared with tumor cells from patients without metastatic disease. Serum Cu concentration in canine OS versus normal canine patients demonstrated similar trends. Dis demonstrated selective cytotoxicity compared with human multipotential stromal cells (MSCs): Dis-treated OS cells demonstrated increased apoptosis, whereas MSCs did not. CuCl2 combined with Dis and low-dose doxorubicin resulted in a superior cytotoxic effect in both SaOS2 and LM7 cell lines. In summary, ALDH gene expression and Cu levels are altered between low and highly metastatic human OS cells, canine samples, and patient tumors. Our findings support the feasibility of a repurposed drug strategy for Dis and Cu in combination with low-dose anthracycline to specifically target metastatic OS cells.

Avenues of research in dietary interventions to target tumor metabolism in osteosarcoma.

Osteosarcoma (OS) is the most frequent primary bone cancer, affecting mostly children and adolescents. Although much progress has been made throughout the years towards treating primary OS, the 5-year survival rate for metastatic OS has remained at only 20% for the last 30 years. Therefore, more efficient treatments are needed. Recent studies have shown that tumor metabolism displays a unique behavior, and plays important roles in tumor growth and metastasis, making it an attractive potential target for novel therapies. While normal cells typically fuel the oxidative phosphorylation (OXPHOS) pathway with the products of glycolysis, cancer cells acquire a plastic metabolism, uncoupling these two pathways. This allows them to obtain building blocks for proliferation from glycolytic intermediates and ATP from OXPHOS. One way to target the metabolism of cancer cells is through dietary interventions. However, while some diets have shown anticancer effects against certain tumor types in preclinical studies, as of yet none have been tested to treat OS. Here we review the features of tumor metabolism, in general and about OS, and propose avenues of research in dietary intervention, discussing strategies that could potentially be effective to target OS metabolism.

Activation of creER recombinase in the mouse calvaria induces local recombination without effects on distant skeletal segments.

Conditional creER-mediated gene inactivation or gene induction has emerged as a robust tool for studying gene functions in mouse models of tissue development, homeostasis, and regeneration. Here, we present a method to conditionally induce cre recombination in the mouse calvarial bone while avoiding systemic recombination in distal bones. To test our method, we utilized Prx1creER-egfp;td-Tomato mice and delivered 4-hydroxytamoxifen (4-OHT) to the mouse calvaria, subperiosteally. First, we showed that two calvaria subperiosteal injections of 10 µg of 4-OHT (3.3 mg of 4-OHT/kg of body weight) can induce local recombination as efficiently as two intraperitoneal systemic injections of 200 µg of tamoxifen (70 mg of tamoxifen/kg of body weight). Then, we studied the recombination efficiency of various subperiosteal calvaria dosages and found that two subperiosteal injections of 5 µg 4-OHT (1.65 mg of 4-OHT/kg of body weight) uphold the same recombination efficiency observed with higher dosages. Importantly, the result indicated that the low dosage does not induce significant systemic recombination in remote skeletal tissues. With the proposed local low dosage protocol, the recombination efficiency at the injection site (calvarial bone) reached 94%, while the recombination efficiency at the mandible and the digits was as low as the efficiency measured in control animals.

Outcome of early dental implant placement versus other dental implant placement protocols: A systematic review and meta-analysis.

BACKGROUND: The aim of this systematic review and meta-analysis was to compare the clinical efficacy of the early dental implant placement protocol with immediate and delayed dental implant placement protocols. METHODS: An electronic and manual search of literature was made to identify clinical studies comparing early implant placement with immediate or delayed placement. Data from the included studies were pooled and quantitative analyses were performed for the implant outcomes reported as the number of failed implants (primary outcome variable) and for changes in peri-implant marginal bone level, peri-implant probing depth, and peri-implant soft tissue level (secondary outcome variables). RESULTS: Twelve studies met the inclusion criteria. Significant difference in risk of implant failure was found neither between the early and immediate placement protocols (risk difference = -0.018; 95% confidence interval [CI] = -0.06, 0.025; P = 0.416) nor between early and delayed placement protocols (risk difference = -0.008; 95% CI = -0.044, 0.028; P = 0.670). Pooled data of changes in peri-implant marginal bone level demonstrated significantly less marginal bone loss for implants placed using the early placement protocol compared with those placed in fresh extraction sockets (P = 0.001; weighted mean difference = -0.14 mm; 95% CI = -0.22, -0.05). No significant differences were found between the protocols for the other variables. CONCLUSIONS: The available evidence supports the clinical efficacy of the early implant placement protocol. Present findings indicate that the early implant placement protocol results in implant outcomes similar to immediate and delayed placement protocols and a superior stability of peri-implant hard tissue compared with immediate implant placement.

Postnatal Calvarial Skeletal Stem Cells Expressing PRX1 Reside Exclusively in the Calvarial Sutures and Are Required for Bone Regeneration.

Post-natal skeletal stem cells expressing PRX1 (pnPRX1+) have been identified in the calvaria and in the axial skeleton. Here we characterize the location and functional capacity of the calvarial pnPRX1+ cells. We found that pnPRX1+ reside exclusively in the calvarial suture niche and decrease in number with age. They are distinct from preosteoblasts and osteoblasts of the sutures, respond to WNT signaling in vitro and in vivo by differentiating into osteoblasts, and, upon heterotopic transplantation, are able to regenerate bone. Diphtheria toxin A (DTA)-mediated lineage ablation of pnPRX1+ cells and suturectomy perturb regeneration of calvarial bone defects and confirm that pnPRX1+ cells of the sutures are required for bone regeneration. Orthotopic transplantation of sutures with traceable pnPRX1+ cells into wild-type animals shows that pnPRX1+ cells of the suture contribute to calvarial bone defect regeneration. DTA-mediated lineage ablation of pnPRX1+ does not, however, interfere with calvarial development.

Simultaneous Ridge Augmentation and Accelerated Molar Uprighting for Implant Site Development: Two Case Reports with a Split-Mouth Design.

Well-coordinated interdisciplinary dental treatments provide the best esthetic, functional, and long-term results for patients. However, the length of such treatment, which may involve orthodontics, ridge augmentation, and dental implants, often deters patients from pursuing them. The two case reports presented here aim to present the advantage of simultaneous orthodontic molar uprighting and ridge augmentation procedures for future implant site development. Selective decortication of the alveolar bone, performed simultaneously with bone grafting, can accelerate the tooth uprighting process and synergistically reduce treatment duration. Two cases with bilaterally missing mandibular first molars were treated with this approach. In both patients, surgically accelerated uprighting of molars occurred 1.6 times faster than the contralateral site, where no surgery was performed. Additionally, ridge augmentation was successfully achieved with 2.5 to 5 mm of horizontal bone gain during the molar uprighting process.

Potential for Stem Cell-Based Periodontal Therapy.

Periodontal diseases are highly prevalent and are linked to several systemic diseases. The goal of periodontal treatment is to halt the progression of the disease and regenerate the damaged tissue. However, achieving complete and functional periodontal regeneration is challenging because the periodontium is a complex apparatus composed of different tissues, including bone, cementum, and periodontal ligament. Stem cells may represent an effective therapeutic tool for periodontal regeneration due to their plasticity and their ability to regenerate different tissues. This review presents and critically analyzes the available information on stem cell-based therapy for the regeneration of periodontal tissues and suggests new avenues for the development of more effective therapeutic protocols.

A novel technique for guided bone regeneration using platelet-rich plasma and osteogenic progenitor cells: Literature-based rationale and case report.

Achieving predictable guided bone regeneration in critical size defects for future endosseous dental implant therapy poses a great challenge to clinicians. A novel technique utilizing autogenous osteogenic progenitor cells, calcium sulfate activated platelet-rich plasma in addition to particulate allograft was successfully used to augment a severely deficient maxillary anterior edentulous ridge. After 6 months of healing, satisfactory radiographic and clinical bone gain was noted with significant increase in alveolar ridge width. Endosseous implants were placed and restored successfully. The techniques with underlying clinical and biologic rationales are presented and discussed in this report.

Effects on Bone Repair of Osteotomy With Drills or With Erbium, Chromium: Yttrium-Scandium-Gallium-Garnet Laser: Histomorphometric and Immunohistochemical Study.

BACKGROUND: The erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser has been widely used in the dental clinic; however, few studies have demonstrated the advantages of the use of this laser for bone osteotomies. The purpose of this study is to evaluate and compare the bone repair process of defects generated by the Er,Cr:YSGG laser or conventional drills. METHODS: Ninety-six rats were divided into two groups of 48 animals (drill group and laser group). After surgical exposure of the right tibia, the animals were subjected to a 2-mm-diameter osteotomy created by conventional drills (drill group) or by the Er,Cr:YSGG laser (laser group). The animals were sacrificed 0, 3, 7, 14, 30, and 60 days after the creation of the defect, and histologic sections were obtained and used for histomorphometric and immunohistochemical analyses for the detection of osteocalcin, osteoprotegerin, receptor activator of nuclear factor κ-B ligand, vascular endothelial growth factor, and caspase-3. RESULTS: The osteotomy with the drill produced well-delimited and smooth walls, whereas the osteotomies in the laser group were irregular and presented an amorphous basophilic line and bone necrosis that was slowly resorbed during the repair process. Despite these characteristics, bone repair was similar between groups at various time points, and, at 60 days, the defects in both groups were completely repaired by newly formed bone. CONCLUSION: The repair process of osteotomies created by the Er,Cr:YSGG laser, despite producing thermal damage to bone tissue, is comparable to that with conventional drills.

Dynamics of bone healing after osteotomy with piezosurgery or conventional drilling - histomorphometrical, immunohistochemical, and molecular analysis.

BACKGROUND: Piezosurgery is an osteotomy system used in medical and dental surgery. Many studies have proven clinical advantages of piezosurgery in terms of quality of cut, maneuverability, ease of use, and safety. However, few investigations have tested its superiority over the traditional osteotomy systems in terms of dynamics of bone healing. Therefore, the aim of this study was to evaluate the dynamics of bone healing after osteotomies with piezosurgery and to compare them with those associated to traditional bone drilling. METHODS: One hundred and ten rats were divided into two groups with 55 animals each. The animals were anesthetized and the tibiae were surgically exposed to create defects 2 mm in diameter by using piezosurgery (Piezo group) and conventional drilling (Drill group). Animals were sacrificed at 3, 7, 14, 30 and 60 days post-surgery. Bone samples were collected and processed for histological, histomorphometrical, immunohistochemical, and molecular analysis. The histological analysis was performed at all time points (n = 8) whereas the histomorphometrical analysis was performed at 7, 14, 30 and 60 days post-surgery (n = 8). The immunolabeling was performed to detect Vascular Endothelial Growth Factor (VEGF), Caspase-3 (CAS-3), Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL), and Osteocalcin (OC) at 3, 7, and 14 days (n = 3). For the molecular analysis, animals were sacrificed at 3, 7 and 14 days, total RNA was collected, and quantification of the expression of 21 genes related to BMP signaling, Wnt signaling, inflammation, osteogenenic and apoptotic pathways was performed by qRT-PCR (n = 5). RESULTS: Histologically and histomorphometrically, bone healing was similar in both groups with the exception of a slightly higher amount of newly formed bone observed at 30 days after piezosurgery (p < 0.05). Immunohistochemical and qRT-PCR analyses didn't detect significant differences in expression of all the proteins and most of the genes tested. CONCLUSIONS: Based on the results of our study we conclude that in a rat tibial bone defect model the bone healing dynamics after piezosurgery are comparable to those observed with conventional drilling.

Localized periodontal defect associated with unusual furcation involvement on a mandibular incisor.

A localized periodontal defect associated with an unusual furcation on a mandibular central incisor and its treatment sequences are presented. A 54-year-old woman presented with a persistent localized periodontal defect, which was not responsive to nonsurgical periodontal therapy. An exploratory surgery revealed complete through and through furcation involvement on the right mandibular central incisor, resulting in a three-walled infrabony defect. Following thorough mechanical debridement and root planing, the infrabony aspect of the defect was grafted with freeze-dried bone allograft combined with the recombinant human platelet-derived growth factor-BB. The flaps were positioned to enable patient's home care and professional dental cleaning. Follow-up examination revealed uneventful healing and resolution of the infrabony periodontal defect. Clinicians should be aware of this unusual condition and consider it as a potential etiology when dealing with a persistent localized periodontal defect in the mandibular anterior sextant, which may not respond to the conventional nonsurgical periodontal therapy. Eliminating the active disease as well as enabling careful maintenance and oral hygiene may prevent further deterioration and result in a stable long-term outcome preserving the compromised tooth.

Future approaches in periodontal regeneration: gene therapy, stem cells, and RNA interference.

Periodontal disease is a major public health issue and the development of effective therapies to treat the disease and regenerate periodontal tissue is an important goal of today's medicine. This article highlights recent scientific advancements in gene therapy, stem cell biology, and RNA interference with the intent of identifying their potential in periodontal tissue regeneration. Results from basic research, preclinical, and clinical studies indicate that these fields of research may soon contribute to more effective regenerative therapies for periodontal disease.

The use of platelet-rich plasma in bone reconstruction therapy.

The use of platelet-rich plasma (PRP) in bone reconstruction therapy was introduced in the late 1990s. Since then, many scientists and clinicians have employed it in orthopaedic and oral surgeries. Unfortunately, studies that analyze the use of PRP are somewhat controversial as some conclude that the use of PRP may favor bone regeneration and others conclude that the use of PRP is irrelevant. By listing and analyzing the biological effect that each factor released by the activated platelets can have in bone regeneration, the present review answers the question of why PRP may be useful in bone reconstruction therapy. Subsequently, by examining the studies that have both successfully and unsuccessfully utilized PRP, it suggests how PRP might be used in order to achieve successful results in orthopaedic and dental bone reconstruction surgeries.

Modulation of MUC7 mucin expression by exogenous factors in airway cells in vitro and in vivo.

The human MUC7 gene encodes a low-molecular-mass mucin that participates in the maintenance of healthy epithelium in the oral cavity, and possibly in respiratory tracts, by promoting the clearance of various bacteria. We examined whether MUC7 gene is expressed in primary normal human tracheobronchial epithelial cells and whether the expression is modulated by exogenous factors. By assessing MUC7 transcripts, we found that the MUC7 gene was induced by culturing the normal human tracheobronchial epithelial cells at the air-liquid interface, in which the cells were well differentiated. When the cells were treated with a panel of cytokines (IL-1beta, IL-4, IL-13, and TNF-alpha), epidermal growth factor, or a bacterial product (Pseudomonas aeruginosa lipopolysaccharide [LPS]), MUC7 transcripts and glycoprotein products were increased 1.7- to 3.2-fold. The effect of LPS on MUC7 gene expression was also studied in the airway tissues of MUC7 gene transgenic mice. In the in vitro cultured trachea and lung explants, the LPS-treated tissues showed over 2-fold increased levels of MUC7 mRNA compared with the untreated specimens. These results were confirmed by in vivo studies using the lungs and tracheas harvested from the transgenic mice irritated by LPS through the tracheal instillation. By immunohistochemistry, MUC7 glycoprotein was localized in tracheal submucosa within the serous cells. Upon LPS stimulation, the overexpressed MUC7 remains confined to the serous glands. In the lungs, MUC7 seems to be expressed within the respiratory epithelium at the level of the bronchioles. Upon stimulation with LPS, it seems to be overexpressed within the same cells and within the stromal tissue.

Platelet-derived growth factor enhancement of two alloplastic bone matrices.

BACKGROUND: The use of alloplastic matrices that mimic the mineral phase of bone has become a viable alternative to current mainstream therapies in dentistry such as allografts and autogenous grafts. Because alloplastic bone substitutes generally have relatively poor osteogenic properties, analyzing their potential as vehicles to deliver growth factors is an important step in assessing methods to enhance their clinical efficacy. The aim of these studies was to treat beta-tricalcium phosphate (beta-TCP) and calcium sulfate (CaSO(4)) with platelet-derived growth factor (PDGF)-BB to enhance the osteogenic capabilities of these materials. METHODS: In the beta-TCP studies, PDGF-BB adsorption and release were accomplished using (125)I radiolabeled growth factor and non-radioactive human recombinant PDGF at a ratio of 1:300 M. For the adsorption studies, the radiolabeled PDGF-BB/ non-radioactive PDGF solutions with resultant PDGF concentrations of 10(7) and 10(8) M were incubated with beta-TCP from 1 to 120 minutes, and the amount of adsorbed (125)I-PDGF-BB was measured using a gamma counter. Similar adsorption studies were conducted with a 30-minute incubation of beta-TCP with various PDGF concentrations. In vitro release studies were conducted with beta-TCP to which radiolabeled PDGF had been adsorbed as above. Release studies were also conducted with CaSO(4) that was hydrated with the radioactive PDGF solution described above for the TCP studies. In vivo PDGF-BB release from beta-TCP and CaSO(4) was evaluated in a mouse model, where the radioactive PDGF/non-radioactive PDGF-BB treated beta-TCP or CaSO(4) sample was inserted subcutaneously and later removed for radioactive measurement. Proliferation of human osteoblastic cells in the presence of PDGF- treated beta-TCP or CaSO(4) was assessed by (3)H thymidine incorporation. RESULTS: The absorption studies revealed that PDGF-BB was absorbed in a concentration and time-dependent manner to beta-TCP. In the in vitro release studies, approximately 45% of the adsorbed PDGF-BB was released after 10 days. In vivo release from both materials occurred faster than in vitro release. Osteoblastic cells incubated with PDGF-BB-treated matrices showed significantly (P <0.05, ANOVA) greater proliferation than with control matrices alone. CONCLUSION: These experiments demonstrate the feasibility of using PDGF-BB in combination with alloplastic materials such as beta-TCP or CaSO(4) to serve as more effective bone graft materials with enhanced osteogenic properties.