Modified Goldner Trichrome for Non-decalcified Mineralized Tissue Plastinated and Embedded in Resin

SUMMARY: Over time, Goldner's trichrome staining has been essential in paraffin soft tissue research. However, its classic application involves prior decalcification, generating disadvantages in the integrity of the samples and the interpretation of results. This study seeks to overcome the limitations associated with decalcification when applying Goldner's trichrome stain with plastic resins. It focuses on detailed visualization of non-decalcified bone and dental samples in animal models. Samples of jaw and tooth from a dog (Canis familiaris) were used, as well as tibia from a rabbit (Oryctolagus cuniculus) with a titanium dental implant and bone graft substitute. Adjustments were made to the original protocol, including a surface treatment prior to staining. Plastination and inclusion in specific plastic resins were part of the process. The microplastinated and stained samples showed optimal quality for optical microscopy. Those from dogs allowed detailed observation of the tooth-periodontal tissue relationship, while those from rabbits revealed a clear differentiation between mineralized and osteoid bone tissue. The staining made it easy to examine the precise interface between soft tissues, bone graft, and implant. The successful adaptation of Goldner's trichrome stain to specimens in plastic resins represents a significant advance in histological investigation of hard tissues. This methodology stands out as an effective tool to evaluate implants and biomaterials in animal models, providing detailed visualization without compromising the integrity of the samples. The combination of histochemistry and plastic resins offers a valuable alternative for microanatomical studies, opening new possibilities in hard tissue research and evaluation of bone structures.

A lo largo del tiempo, la tinción tricrómica de Goldner ha sido esencial en la investigación de tejidos blandos en parafina. Sin embargo, su aplicación clásica conlleva la descalcificación previa, generando desventajas en la integridad de las muestras y la interpretación de resultados. Este estudio busca superar las limitaciones asociadas con la descalcificación al aplicar la tinción tricrómica de Goldner con resinas plásticas. Se enfoca en visualizar detalladamente muestras óseas y dentales no descalcificadas en modelos animales. Se emplearon muestras de mandíbula y diente de perro (Canis familiaris), así como tibia de conejo (Oryctolagus cuniculus) con implante dental de titanio y substituto de injerto óseo. Se realizaron ajustes al protocolo original, incluyendo un tratamiento superficial previo a la tinción. La plastinación y la inclusión en resinas plásticas específicas fueron parte del proceso. Las muestras microplastinadas y teñidas mostraron una calidad óptima para microscopía óptica. Las de perro permitieron la observación detallada de la relación diente-tejido periodontal, mientras que las de conejo revelaron una clara diferenciación entre tejido óseo mineralizado y osteoide. La tinción facilitó examinar la interface precisa entre tejidos blandos, injerto óseo e implante. La adaptación exitosa de la tinción tricrómica de Goldner a muestras en resinas plásticas representa un avance significativo en la investigación histológica de tejidos duros. Esta metodología destaca como una herramienta eficaz para evaluar implantes y biomateriales en modelos animales, brindando una visualización detallada sin comprometer la integridad de las muestras. La combinación de histoquímica y resinas plásticas ofrece una alternativa valiosa para estudios microanatómicos, abriendo nuevas posibilidades en la investigación de tejidos duros y evaluación de estructuras óseas.

Effect of silicon-doped calcium phosphate bone grafting materials on bone regeneration and osteogenic marker expression after implantation in the ovine scapula.

Compared to the currently clinically available bone grafting materials for alveolar ridge augmentation, there is a great demand for bioactive bone substitutes with higher resorbability, which enhance osteogenesis at the same time. This has prompted the development of a silicon-doped rapidly resorbable calcium alkali orthophosphate (Si-CAOP) and silicon-doped ß-tricalcium phosphate (Si-TCP). This study evaluated the effect of these two particulate graft materials as compared to the currently clinically used ß-TCP on bone formation and osteogenic marker expression after 2 weeks, 1, 3, 6, 12, and 18 months of implantation in critical size defects in the sheep scapula. Immunohistochemical analysis of collagen type I, alkaline phosphatase, and osteocalcin expression was performed on resin embedded sections. The bone and particle area fraction and the bone-biomaterial contact were determined histomorphometrically. After 2 weeks and 1 month defects grafted with Si-CAOP displayed a significantly greater bone area fraction, bone-particle-contact, osteogenic marker expression and significantly lower particle area fraction than defects grafted with Si-TCP and TCP. By 3 and 6 months all materials studied mediated excellent defect regeneration with further bone remodeling at 12 and 18 months. Taken together, Si-CAOP induced the most expeditious bone regeneration of critical size defects in the sheep scapula. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 594-614, 2019.

Effect of a Particulate and a Putty-Like Tricalcium Phosphate-Based Bone-grafting Material on Bone Formation, Volume Stability and Osteogenic Marker Expression after Bilateral Sinus Floor Augmentation in Humans.

This study examines the effect of a hyaluronic acid (HyAc) containing tricalcium phosphate putty scaffold material (TCP-P) and of a particulate tricalcium phosphate (TCP-G) graft on bone formation, volume stability and osteogenic marker expression in biopsies sampled 6 months after bilateral sinus floor augmentation (SFA) in 7 patients applying a split-mouth design. 10% autogenous bone chips were added to the grafting material during surgery. The grain size of the TCP granules was 700 to 1400 µm for TCP-G and 125 to 250 µm and 500 to 700 µm (ratio 1:1) for TCP-P. Biopsies were processed for immunohistochemical analysis of resin-embedded sections. Sections were stained for collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Furthermore, the bone area and biomaterial area fraction were determined histomorphometrically. Cone-beam CT data recorded after SFA and 6 months later were used for calculating the graft volume at these two time points. TCP-P displayed more advantageous surgical handling properties and a significantly greater bone area fraction and smaller biomaterial area fraction. This was accompanied by significantly greater expression of Col I and BSP and in osteoblasts and osteoid and a less pronounced reduction in grafting volume with TCP-P. SFA using both types of materials resulted in formation of sufficient bone volume for facilitating stable dental implant placement with all dental implants having been in function without any complications for 6 years. Since TCP-P displayed superior surgical handling properties and greater bone formation than TCP-G, without the HyAc hydrogel matrix having any adverse effect on bone formation or graft volume stability, TCP-P can be regarded as excellent grafting material for SFA in a clinical setting. The greater bone formation observed with TCP-P may be related to the difference in grain size of the TCP granules and/or the addition of the HyAc.

A novel multimodular methodology to investigate external cervical tooth resorption.

AIM: To introduce a multimodular combination of techniques as a novel minimal invasive approach to investigate efficiently and accurately external cervical resorption (ECR). METHODOLOGY: One case of a central incisor with extensive external cervical resorption was selected to demonstrate the potential of a comparative novel study methodology. ECR diagnosis was based on clinical inspection, digital radiography and cone-beam computed tomography (CBCT). After extraction, the tooth was investigated using microfocus computed tomography (micro-CT), nano-CT and hard tissue histology. These techniques were compared for their accuracy and applicability to highlight their advantages and disadvantages. RESULTS: Nano-CT was more effective than micro-CT and CBCT for detailed ex vivo exploration of ECR. The reparative tissue, pericanalar resorption resistant sheet (PRRS), pulp tissue reactions, resorption channels and their interconnection with the periodontal ligament space were accurately visualized by detailed processing and analysis of the nano-CT data set with Dataviewer and CTAn software. Nano-CT analysis provided better insight in the true extent of the resorption, based on quantitative measurements and 3D visualization of the tooth structure. Nano-CT imaging results were similar to hard tissue histology at the mineralized tissue level. To clarify the dynamic phenomenon of reparative tissue formation and substitution of the resorbed tissues, nano-CT needed to be associated with hard tissue histology. CONCLUSION: Nano-CT is a fast and minimal invasive technique for the ex vivo analysis and understanding of ECR and is complementary with hard tissue histology. A combined approach of clinical and CBCT examination, with nano-CT and histological mapping measurements, can provide an ideal platform for future ECR imaging and exploration studies.