Acid Decalcification Research Articles

Biomacromolecules within bivalve shells: Is chitin abundant?

Bivalve shells are inorganic-organic nanocomposites whose material properties outperform their purely inorganic mineral counterparts. Most typically the inorganic phase is a polymorph of CaCO3, while the organic phase contains biopolymers which have been presumed to be chitin and/or proteins. Identifying the biopolymer phase is therefore a crucial step in improving our understanding of design principles relevant to biominerals. In this work we study seven shells; four are examples of nacroprismatic shells (Alathyria jacksoni, Pinctada maxima, Hyriopsis cumingii and Cucumerunio novaehollandiae), one homogeneous (Arctica islandica), and two are crossed lamellar (Callista kingii, Tridacna gigas). Both intact shells, their organic extracts as isolated after decalcification in acid, and the periostracum overlay have been studied by solid-state CP-MAS NMR, FTIR, SEM and chemical analysis. In none of the shells examined in this work do we find a significant contribution to the organic fraction from chitin or its derivatives despite popular models of bivalve biomineralization which assume abundant chitin in the organic fraction of mollusk bivalve shells. In each of the nacroprismatic extracts the 13C NMR spectra represent similar proteinaceous material, Ala and Gly-rich and primarily organized as β-sheets. A different, yet highly conserved protein was found in the periostracum covering each of the three nacreous shells studied. The Arctica islandica shells with homogeneous microstructure contained proteins which do not appear to be silk-like, while in the crossed lamellar shells we extracted too little organic matter to characterize. Statement of SignificanceHydrophobic macromolecules are structural components within the calcareous inorganic matrix of bivalve shells and are responsible for enhanced materials properties of the biominerals. Prevalent models suggest that chitin is such major hydrophobic component. Contrary to that we show that chitin is rare within the hydrophobic biopolymers which primarily consist of proteinaceous matter with structural motifs as silk-like β-sheets, or others yet to be determined. Recognizing that diverse proteinaceous motifs, devoid of abundant chitin, can yield the optimized mechanical properties of bivalve shells is critical both to understand the mechanistic pathways by which they regulate biomineralization and for the design of novel bioinspired materials.

Comparison of decalcifying agents and techniques for human dental tissues

Teeth are among the hardest animal tissues, because they are composed of large amounts of inorganic compounds. Consequently, teeth are difficult to prepare for microscopic examination. Acids and chelating agents traditionally have been used to remove calcium ions. We compared decalcifying agents including strong acids, weak acids, chelating agents, techniques using electric current, agitation and heat. Freshly extracted teeth were fixed and decalcified using formic acid-formalin, formal-nitric acid, formalin-EDTA, Von Ebner’s fluid and Perenyi’s fluid. Three additional techniques including formic acid with agitation, formic acid with heat and formic acid with electric current also were evaluated. Decalcified teeth were evaluated histologically for tissue preservation and staining characteristics. Formic acid with gentle agitation produced the best decalcification overall based on time required for decalcification, ease of sectioning, hard and soft tissue staining and tissue preservation. Our findings support the use of agitation with formic acid decalcification, because it reduces significantly both the time required and the deleterious effects of prolonged immersion.

Effect of Hydrochloric Acid Decalcification on Expression Pattern of Prognostic Markers in Invasive Breast Carcinomas.

In the United States, it is estimated that 100,000 people are living with metastatic breast cancer (BC) with bone representing the most common site of involvement. However, patients with isolated bone metastasis at presentation may have a longer survival. Therapeutic options for BC bone metastases often include systemic anticancer therapy (endocrine, chemotherapy, monoclonal antibodies, and/or other targeted therapies), which is largely dependent on the immunohistochemical (IHC) repertoire of the cancer for the prognostic markers [estrogen (ER) and progesterone receptors (PR), Ki-67, p53, and Her-2/neu] at its osseous metastatic site. Traditionally, specimens obtained from the bone metastasis require decalcification, which may affect the immunoreactivity of these prognostic markers. To the best of our knowledge, limited studies describe the effect of decalcification on immunoexpression of the above-mentioned markers. A detailed illustration of the effect of decalcification on BC specimens in a real-time manner is lacking in the literature. Herein, we sought to determine the impact of decalcification on the IHC expression pattern of the above listed markers on BC tissue following decalcification. After Institutional Review Board approval, sections from the residual tumor specimens were collected prospectively from 15 BC excision specimens and 1 curetting from a BC bone metastasis. The sections (3 to 6 sections/case) for decalcification were collected following routine submission for pathologic evaluation. The sections were subjected to hydrochloric acid (HCl)-based Decal Stat decalcifying solution for 2, 12, 18, and 24 hours in each case. IHC studies for ER, PR, Ki-67, p53, and Her-2/neu were performed on 1 representative section of the regularly processed tumor block and 1 decalcified tumor block from each time point. Scoring of ER and PR were performed according to the Allred scoring system. Scoring of Her-2/neu was performed according to CAP/ASCO guidelines. The tumors comprised 11 grade 3 invasive ductal carcinomas, 2 grade 2 invasive ductal carcinomas, 2 grade 3 invasive lobular carcinoma, and 1 metastatic BC to bone. Nine cases showed Allred score 8 for ER, 1 case showed Allred score 4, 1 case showed Allred score 2, and the remaining 5 were ER negative. For PR, 1 showed Allred score 8, 2 Allred score 7, 4 Allred score 6, 1 each Allred score 5 and score 2 with the remainder negative for PR. Ki-67 ranged from 5% to 95%. Five cases showed p53 overexpression ranging from 35% to 95%. Five cases each showed 3+, 6 cases showed 2+ Her-2/neu, 3 cases showed 1+ Her-2/neu, and the remaining 2 were negative. All specimens demonstrated decline in ER, PR, Ki-67, and p53 immunoreactivity after 2 hours of decalcification, with additional decline up to 24 hours. The most significant declines in immunoreactivity occurred with Ki-67 and p53. Most of the Her-2/neu cases with an equivocal score declined to zero after 24 hours of decalcification. However, 3 out of 11 cases showing Her-2/neu overexpression remained at the baseline scoring even after extended (24 h) decalcification. Our results demonstrated that the decalcification process affects the immunoreactivity of the prognostic BC markers. There is progressive loss of reactivity at 2 hours and beyond for markers with lower degrees of expression. In addition, heterogeneity in marker distribution progressed from diffuse to more focal beyond 1 hour.

Rotary ultrasonic drilling on bone: A novel technique to put an end to thermal injury to bone.

Bone drilling is common in orthopedic procedures and the heat produced during conventional experimental drilling often exceeds critical temperature of 47 °C and induces thermal osteonecrosis. The osteonecrosis may be the reason for impaired healing, early loosening and implant failure. This study was undertaken to control the temperature rise by interrupted cutting and reduced friction effects at the interface of drill tool and the bone surface. In this work, rotary ultrasonic drilling technique with diamond abrasive particles coated on the hollow drill tool without any internal or external cooling assistance was used. Experiments were performed at room temperature on the mid-diaphysis sections of fresh pig bones, which were harvested immediately after sacrifice of the animal. Both rotary ultrasonic drilling on bone and conventional surgical drilling on bone were performed in a five set of experiments on each process using identical constant process parameters. The maximum temperature of each trial was recorded by K-type thermocouple device. Ethylenediaminetetraacetic acid decalcification was done for microscopic examination of bone. In this comparative procedure, rotary ultrasonic drilling on bone produced much lower temperature, that is, 40.2 °C ± 0.4 °C and 40.3 °C ± 0.2 °C as compared to that of conventional surgical drilling on bone, that is, 74.9 °C ± 0.8 °C and 74.9 °C ± 0.6 °C with respect to thermocouples fixed at first and second position, respectively. The conventional surgical drilling on bone specimens revealed gross tissue burn, microscopic evidence of thermal osteonecrosis and tissue injury in the form of cracks due to the generated force during drilling. But our novel technique showed no such features. Rotary ultrasonic drilling on bone technique is robust and superior to other methods for drilling as it induces no thermal osteonecrosis and does not damage the bone by generating undue forces during drilling.

Mutational profiling of bone metastases from lung adenocarcinoma: results of a prospective study (POUMOS-TEC).

Targeted therapies have improved patient survival in metastatic lung adenocarcinoma. Molecular diagnosis is a key element to identify oncogenic drivers predicting the efficacy of these agents. In stage IV patients, histopathological diagnosis is often performed on bone metastases biopsy, but routine procedure of decalcification may alter DNA quality for subsequent molecular tests. We set up a procedure to perform molecular analyses on bone metastasis and describe the results of mutational profiling. POUMOS-TEC is a prospective study conducted in stage IV lung adenocarcinomas. Bone metastasis specimens from surgery and CT-scan guided biopsies were sent fresh for immediate formalin-fixation. Decalcification was performed, only when necessary, using EDTA. Controls were processed with acid decalcification. DNA extraction was performed after laser microdissection. Mutational profiling of oncogenic drivers was conducted as recommended by the French National Cancer Institute. Diagnosis efficiency of the computed tomography (CT)-scan guided biopsy process was assessed. Among 177 collected bone metastases specimens, 49 came from lung adenocarcinomas. Specimens processed with no decalcification or EDTA (n=45) provided high-quality DNA. Molecular profiling was performed in 44/45 (98%) of cases. The results of the whole panel of oncogenic drivers (EGFR, KRAS, BRAF, PIK3CA, HER2 and ALK) were obtained in 41/45 (91%) of cases. A mutation was observed in 50% of cases including 32% of KRAS and 14% of epidermal growth factor receptor (EGFR) mutations. CT-scan biopsy efficiency rate was 96%. We demonstrated the feasibility to routinely conduct mutational profiling on bone metastases biopsies. We observed a higher rate of EGFR mutations (+42%) in comparison with the average rate of all stage IV lung adenocarcinomas. This procedure is a new step toward the goal of personalized medicine to treat lung cancers and other osteophilic tumors.

Comparative evaluation of a dentifrice containing calcium sodium phosphosilicate to a dentifrice containing potassium nitrate for dentinal hypersensitivity: A clinical study.

Background:Calcium sodium phosphosilicate is a recently introduced desensitizing agent which acts by occluding the dentinal tubules and also resists acid decalcification. This study was designed to assess the efficacy of a new toothpaste containing 5% calcium sodium phosphosilicate for the treatment of dentinal hypersensitivity and also compare it with 5% potassium nitrate.Materials and Methods:Sixty patients with the chief complaint of dentinal hypersensitivity were enrolled and randomly divided into two groups. The visual analog scale (VAS) scores were taken for water and air stimuli at baseline, 3 weeks after usage of the respective toothpaste, and 3 weeks after discontinuation of the respective toothpaste.Results:Both the groups showed reduction in hypersensitivity scores at 3 weeks and 6 weeks for air stimulus and cold water. The calcium sodium phosphosilicate group, however, showed significantly reduction in hypersensitivity compared to the potassium nitrate group at any time point for both measures of hypersensitivity.Conclusion:The 5% calcium sodium phosphosilicate group showed immense reduction in dentinal hypersensitivity symptoms. The 5% calcium sodium phosphosilicate showed prolonged effects even after discontinuation as compared to 5% potassium nitrate, due to its dentinal tubular occlusion property.

Comparison of standard laminectomy with an optimized ejection method for the removal of spinal cords from rats and mice

For researchers seeking to collect spinal cord samples from mice and rats while avoiding acid decalcification, few options are available. Laminectomy is the standard method which yields high quality samples, yet is time consuming and technically difficult. Ejection of the cord from the vertebral column is another technique commonly used; however, the literature suggests that this method can damage the spinal tissues and is typically avoided when histology of samples is the desired endpoint. Here, we describe an optimized method for ejection of spinal cords from rats and mice, and compare histological quality of these samples with those collected via laminectomy. Our results show that ejection can yield high quality spinal cord samples and may be suitable for use as an alternative to laminectomy.

Diffusion of aniline blue injected into the thyroarytenoid muscle as a proxy for botulinum toxin injection: an experimental study in cadaver larynges.

Summary Introduction: Endolaryngeal injection of botulinum toxin into the thyroarytenoid (TA) muscle is one of the methods for treatment of focal laryngeal dystonia. However, after treatment, there is variation in laryngeal configuration as well as the side effects reported by patients. As a consequence of the functional variability of results, it was hypothesized that botulinum toxin diffuses beyond the limits of the muscle into which it is injected. Objectives: After injection of botulinum toxin into the TA muscle for the treatment of focal laryngeal dystonia, patients differ in terms of laryngeal configuration and side effects. We hypothesized that this toxin diffuses from the target muscle to adjacent muscles. Method: The TA muscles of 18 cadaver larynges were injected with aniline blue (0.2 mL). After fixation in formaldehyde and nitric acid decalcification, the larynges were sectioned in the coronal plane and the intrinsic muscles were analyzed. Results: We found diffusion of aniline blue to the lateral cricoarytenoid muscle, cricothyroid muscle, and posterior cricoarytenoid muscle in 94.3%, 42.9%, and 8.6% of the cases, respectively. In terms of the degree of diffusion to adjacent muscles, we found no differences related to the size (height and width) of the TA muscle or to gender. Conclusions: Our findings suggest that diffusion of botulinum toxin from its injection site in the TA muscle to the lateral cricoarytenoid muscle is likely in most cases. On the other hand, diffusion to the cricothyroid muscle occurs in approximately half of cases and diffusion to the posterior cricoarytenoid muscle occurs in very few cases.

Comparison of routine decalcification methods with microwave decalcification of bone and teeth

Introduction:The microwave oven has been used quite often for tissue processing, but there are very few studies describing its use in decalcification of bone or teeth. In this study we have attempted to decalcify bone and teeth using a microwave oven and compare the process and results with conventional decalcification methods.Aims and objectives:The objectives of the study were to determine and compare routine decalcification with microwave decalcification of bone and teeth using 5% nitric acid, 5% formic acid, and 14% ethylenediaminetetraacetic acid (EDTA) with respect to speed of decalcification, preservation of tissue structure and staining efficacy.Materials and methods:In our study the total sample size used for both routine and microwave decalcification was 30 premolar teeth and 30 pieces of condyles. The three solutions were dilute nitric acid (5%), formic acid (5%), and EDTA (14%). Each set consisting of the same type of premolars and condyles in each of the three decalcifying solutions were used in both manual method and microwave method.Results:The results in the present study confirmed the fact that the microwave method using nitric acid was indeed the fastest decalcifying method needing just about 2 days for condyle and 4 days for premolars, compared with routine decalcification. The results also showed that the overall histological picture was good with EDTA and formic acid irrespective of the methods used. In the routine method, nitric acid gave poor cellular detail when compared with microwave method.Conclusion:With our study we conclude that microwave oven decalcification is faster than routine decalcification irrespective of the decalcifying agents used. The tissue preservation and staining efficacy was good in microwave nitric acid decalcification compared to routine nitric acid decalcification. Both formic acid and EDTA show good tissue preservation and staining efficacy irrespective of the method used.

Methods to quantify components of the excavating sponge Cliona orientalis Thiele, 1900

AbstractThis study applied the loss after combustion (LAC) method and the acid decalcification (ADC) method to quantify different components of an excavating sponge. Samples of dried coral skeleton of Favia sp. invaded by the Indo‐Pacific excavating sponge Cliona orientalis Thiele, 1900 were used. The sponge tissue penetrated the 12‐mm‐thick samples to approximately 10 mm. The average proportional weight of organic matter, siliceous spicules, calcareous substrate and salts in the entire samples was found to be respectively 2.5%, 4.4%, 90.5% and 2.5% of dry weight applying the LAC method, and 2.9%, 5.9%, 89.0% and 2.3% of dry weight applying the ADC method. Respective volumetric proportions of the organic matter, spicules, substrate and salts were then calculated to be 6.4%, 5.5%, 85.2% and 3.0% of volume with the LAC method, and 7.4%, 7.2%, 82.7% and 2.7% of volume with the ADC method. The LAC method showed low variability of data and is simple and fast and therefore is recommended. The ADC method generated very similar results to the LAC method. However, due to the handling involved in the ADC method, more than half of the spicules may be lost and the method is therefore not recommended unless careful data corrections are considered. In addition, the buoyant weight method was used to quantify actual substrate weight in the fresh sponge‐substrate samples. This method was found to be at least 97% effective, revealing that buoyant weights can potentially be used to quantify bioerosion rates of excavating sponges. To our knowledge, this is the first study to systemically quantify organic and inorganic components of an excavating sponge and its calcareous substrate, providing improved standard methods for future studies.

Application of Trichloroacetic Acid-Acetone Precipitation Method for Protein Extraction in Bone Tissue

To explore the feasibility and efficiency of extracting protein from bone tissue using trichloroacetic acid (TCA)-acetone precipitation method. Hydrochloric acid (HCL) decalcification method and TCA-acetone precipitation method were separately used for bone protein extraction. The efficiencies of these two methods were compared. TCA-acetone precipitation method had significantly higher extraction efficiency. Compared with HCL decalcification method, it had less pollution from bone marrow hematopoietic tissue. Protein band distribution was similar between these two methods. TCA-acetone precipitation method is useful for bone proteomics research.

Anatomic site variability in rat skeletal uptake and desorption of fluorescently labeled bisphosphonate.

Bisphosphonates commonly used to treat osteoporosis, Paget's disease, multiple myeloma, hypercalcemia of malignancy and osteolytic lesions of cancer metastasis have been associated with bisphosphonate-associated jaw osteonecrosis (BJON). The underlying pathogenesis of BJON is unclear, but disproportionate bisphosphonate concentration in the jaw has been proposed as one potential etiological factor. This study tested the hypothesis that skeletal biodistribution of intravenous bisphosphonate is anatomic site-dependent in a rat model system. Fluorescently labeled pamidronate was injected intravenously in athymic rats of equal weights followed by in vivo whole body fluorimetry, ex vivo optical imaging of oral, axial, and appendicular bones and ethylenediaminetetraacetic acid bone decalcification to assess hydroxyapatite-bound bisphosphonate. Bisphosphonate uptake and bisphosphonate released per unit calcium were similar in oral and appendicular bones but lower than those in axial bones. Hydroxyapatite-bound bisphosphonate liberated by sequential acid decalcification was the highest in oral, relative to axial and appendicular bones (P < 0.05). This study demonstrates regional differences in uptake and release of bisphosphonate from oral, axial, and appendicular bones of immune deficient rats.

Ethylene Diamine Tetra Acetic Acid (EDTA) Decalcification of Paediatric Bone Marrow Trephines In a Diagnostic Laboratory

AbstractAbstract 2566 Background:In paediatric patients with haematological disorders such as acute lymphoblastic leukaemia (ALL), bone marrow aspiration is sometimes difficult to obtain and bone marrow trephine biopsy (BMTB) is a valuable source of material. In a diagnostic laboratory, the turnaround time is critical for a bone marrow trephine to be decalcified, processed and embedded. In our laboratory, 48 hours was routinely required from the time the bone marrow was performed until the sections were ready for reporting. A hydrochloric acid-EDTA (Ethylene-Diamine-Tetra-Acetic acid) decalcifying solution was used for 4 hours but rendered the trephines unsuitable for special studies such as Fluorescence in Situ Hybridisation (FISH). Aim:To evaluate an alternative decalcification method which preserved the ability to perform FISH on formalin-fixed paraffin-embedded (FFPE) tissue without compromising the turnaround time as a laboratory quality improvement measure. Method (EDTA decalcification):Following overnight formalin fixation, the BMTB was decalcified in a solution containing 20% EDTA with continuous stirring for 7.5 hours. The 20% EDTA, pH 7.1 stock solution was prepared by adding Ammonium Hydroxide (25%, concentrated ammonia) (Merck) to distilled water. EDTA disodium salt (372.24; Ajax Finechem) was added and the pH adjusted to pH 7.1 using concentrated ammonia. BM trephines were then processed routinely, embedded in paraffin and 4μm sections were mounted on Super Frost Plus slides. Haematoxylin-Eosin (H&E) staining, Silver Nitrate staining for Reticulin was performed on all slides and Immunocytochemistry, Immunofluorescence and FISH on selected slides. Patient Characteristics:20 trephines from 15 patients underwent 7.5 hour EDTA decalcification. The diagnosis in 9 patients was Precursor-B ALL while one each had T-Cell ALL, Acute Myeloid Leukaemia, Hodgkin's disease, Refractory Anaemia, Drug Induced Anaemia and Idiopathic Thrombocytopenic Purpura. The mean age was 9.3 years (range 1.9–16.7years) and the mean trephine length was 12.8mm (range 6–21mm). Results:100% decalcification was achieved in 18 trephines while in 2 trephines 95% decalcification was achieved on morphological examination. The turnaround time was 48 hours. The quality of H&E, reticulin stain, immunohistochemistry and immunofluorescence was maintained and FISH was successful on these FFPE BMTB tissues. This has lead to incorporation of this method for routine use in our laboratory. Conclusion:20% EDTA decalcification of paediatric BMTB specimens is feasible without affecting the quality of histological preparations or turnaround time. The main advantage of the EDTA decalcification process is that the tissue is amenable to FISH analysis should it be required. Disclosures:No relevant conflicts of interest to declare.

A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling

In this study, we investigated a clinically relevant model of in vivo ectopic bone formation utilizing human periosteum derived cells (HPDCs) seeded in a Collagraft™ carrier and explored the mechanisms by which this process is driven. Bone formation occurred after eight weeks when a minimum of one million HPDCs was loaded on Collagraft™ carriers and implanted subcutaneously in NMRI nu/nu mice. De novo bone matrix, mainly secreted by the HPDCs, was found juxta-proximal of the calcium phosphate (CaP) granules suggesting that CaP may have triggered the ‘osteoinductive program’. Indeed, removal of the CaP granules by ethylenediaminetetraacetic acid decalcification prior to cell seeding and implantation resulted in loss of bone formation. In addition, inhibition of endogenous bone morphogenetic protein and Wnt signalling by overexpression of the secreted antagonists Noggin and Frzb, respectively, also abrogated osteoinduction. Proliferation of the engrafted HPDCs was strongly reduced in the decalcified scaffolds or when seeded with adenovirus-Noggin/Frzb transduced HPDCs indicating that cell division of the engrafted HPDCs is required for the direct bone formation cascade. These data suggest that this model of bone formation is similar to that observed during physiological intramembranous bone development and may be of importance when investigating tissue engineering strategies.

A sensitive mutation‐specific screening technique for GNAS1 mutations in cases of fibrous dysplasia: the first report of a codon 227 mutation in bone

To report on the mutation-specific restriction enzyme digest (MSRED) method using paraffin-embedded tissue as a means of detecting GNAS1 mutations in fibrous dysplasia (FD), and to determine if any of the reported GNAS1 mutations in endocrine neoplasms, not previously documented in FD, can be found in FD. Sixty-seven cases of extragnathic FD were analysed as two groups, 1997-2002 and 2003-06, chosen because tissue fixation and decalcification methods were more accurately recorded in the latter. MSRED revealed that between 2003 and 2006, 93% of 28 'in house' extragnathic cases harboured a GNAS1 mutation, compared with 75% of 32 cases before 2003. Fixation times of no more than 48 h and decalcification in ethylenediamine tetraacetic acid gave the best results. Of the 56 mutations detected (five gnathic, 51 extragnathic), 32 (57%) were R201H, 21 (38%) were R201C and three (5%) were Q227L. Two Q227L extragnathic cases had unusual clinical/radiological findings. No mutations were detected in osteofibrous dysplasia. Detection of GNAS1 mutations by MSRED is a valuable adjunct to the histopathological diagnosis of FD. This is the first report of a Q227L mutation in FD, although it has been previously documented in pituitary adenoma.

Stapes Surgery

Hypothesis: The aim of this study was to examine the long process of the incus in respect of its shape and its dimensions at the site of the attachment of a stapes prosthesis. Background: One of the complications in stapes surgery is the erosion of the long process of the incus at the site of the attachment of the prosthesis, resulting in a fluctuating conductive hearing loss. Knowing the dimensions of the attachment site of the prosthesis at the long process of the incus will make it possible to optimize the size of the prosthesis loop. Methods: The incus was obtained from 11 patients who had undergone middle ear surgery for hearing improvement or cholesteatoma removal. The ossicles were kept in 4% paraformaldehyde and were processed for histological examination after decalcification in ethylenediamine tetra-acetic acid. The 5-μm slices were stained with toluidine blue and examined in the light microscope. Using digitized video images of the histological slices, the diameters, circumference, and surface of the specimens were determined. Results: The diameter of the long process of the incus at 1.4 ± 0.28 mm from the tip, which is the average site of prosthesis attachment, showed an oval shape with a minimum diameter of 0.66 ± 0.05 mm, a maximum diameter of 0.81 ± 0.1 mm, and a circumference of 2.46 ± 0.23 mm. Conclusion: The loop of a stapes prosthesis should have the following dimensions: diameter 0.9 mm, loop length of 2.2 mm, and opening of loop 0.7 mm. However, the material of the prosthesis and its malleability are also important factors.

Stapes Surgery

The aim of this study was to examine the long process of the incus in respect of its shape and its dimensions at the site of the attachment of a stapes prosthesis. One of the complications in stapes surgery is the erosion of the long process of the incus at the site of the attachment of the prosthesis, resulting in a fluctuating conductive hearing loss. Knowing the dimensions of the attachment site of the prosthesis at the long process of the incus will make it possible to optimize the size of the prosthesis loop. The incus was obtained from 11 patients who had undergone middle ear surgery for hearing improvement or cholesteatoma removal. The ossicles were kept in 4% paraformaldehyde and were processed for histological examination after decalcification in ethylenediamine tetra-acetic acid. The 5-microm slices were stained with toluidine blue and examined in the light microscope. Using digitized video images of the histological slices, the diameters, circumference, and surface of the specimens were determined. The diameter of the long process of the incus at 1.4+/-0.28 mm from the tip, which is the average site of prosthesis attachment, showed an oval shape with a minimum diameter of 0.66+/-0.05 mm, a maximum diameter of 0.81+/-0.1 mm, and a circumference of 2.46+/-0.23 mm. The loop of a stapes prosthesis should have the following dimensions: diameter 0.9 mm, loop length of 2.2 mm, and opening of loop 0.7 mm. However, the material of the prosthesis and its malleability are also important factors.

Optimization of Histoprocessing Methods to Detect Glycosaminoglycan, Collagen Type II, and Collagen Type I in Decalcified Rabbit Osteochondral Sections

Rabbit models frequently are used as a small animal cartilage repair model, where hyaline articular cartilage repair can be identified by the presence of type II collagen, the absence of type I collagen, and an abundant glycosaminoglycan component (GAG). Staining methods for these three extracellular matrix components should therefore function in the same sample after histologic processing. The goal of the current study was to optimize histoprocessing techniques to maximally retain GAG, while still enabling the immunodetection of both collagen type II and collagen type I in decalcified rabbit osteochondral sections. We compared four different fixation methods and two different decalcification procedures in both paraffin and cryostat sections using a histological grading scale for five tissue characteristics, including chondrocyte morphology, bone marrow structure, cartilage GAG staining with Safranin O, cartilage immunostaining for type II collagen, and bone matrix immunostaining for type I collagen. We found that addition of ruthenium hexaammine trichloride to the fixation solution provided the best retention of chondrocyte morphology. However, ruthenium hexaammine trichloride interfered with immunodetection of type II collagen, an effect not observed for any of the other fixation conditions. Additionally, paraffin embedding was found to suppress immunodetection of type I collagen in bone matrix compared with cryosectioning. Nonetheless, bone marrow structure was better preserved in paraffin sections. Taken together, our results show that fixation in formalin or paraformaldehyde, followed by decalcification in acid with trace fixative, and then cryosectioning permitted 1) adequate preservation of chondrocyte morphology, 2) Safranin O staining of GAG in cartilage matrix, 3) immunostaining of type II collagen in cartilage matrix, and 4) immunostaining of type I collagen in bone matrix. (The J Histotechnol 28:165, 2005)Submitted March 28, 2005; accepted July 7, 2005

Optimization of Histoprocessing Methods to Detect Glycosaminoglycan, Collagen Type II, and Collagen Type I in Decalcified Rabbit Osteochondral Sections

Rabbit models frequently are used as a small animal cartilage repair model, where hyaline articular cartilage repair can be identified by the presence of type II collagen, the absence of type I collagen, and an abundant glycosaminoglycan component (GAG). Staining methods for these three extracellular matrix components should therefore function in the same sample after histologic processing. The goal of the current study was to optimize histoprocessing techniques to maximally retain GAG, while still enabling the immunodetection of both collagen type II and collagen type I in decalcified rabbit osteochondral sections. We compared four different fixation methods and two different decalcification procedures in both paraffin and cryostat sections using a histological grading scale for five tissue characteristics, including chondrocyte morphology, bone marrow structure, cartilage GAG staining with Safranin O, cartilage immunostaining for type II collagen, and bone matrix immunostaining for type I collagen. We found that addition of ruthenium hexaammine trichloride to the fixation solution provided the best retention of chondrocyte morphology. However, ruthenium hexaammine trichloride interfered with immunodetection of type II collagen, an effect not observed for any of the other fixation conditions. Additionally, paraffin embedding was found to suppress immunodetection of type I collagen in bone matrix compared with cryosectioning. Nonetheless, bone marrow structure was better preserved in paraffin sections. Taken together, our results show that fixation in formalin or paraformaldehyde, followed by decalcification in acid with trace fixative, and then cryosectioning permitted 1) adequate preservation of chondrocyte morphology, 2) Safranin O staining of GAG in cartilage matrix, 3) immunostaining of type II collagen in cartilage matrix, and 4) immunostaining of type I collagen in bone matrix. (The J Histotechnol 28:165, 2005)Submitted March 28, 2005; accepted July 7, 2005

Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer.

Production of paraffin-section material from tissue samples that contain bone requires decalcification. Techniques such as acidic decalcification or EDTA chelation are suitable methods. Acid decalcification is generally quicker than EDTA chelation but studies have suggested that it may result in hydrolysis of DNA. Here we show that limited acid decalcification (less than 24 hr) in 5% formic acid can preserve DNA sufficient for fluorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH) and that prolonged 10% formic acid decalcification results in failure of FISH and only limited retrieval of DNA for CGH studies.