Study on injectable calcium sulfate bone cement rich in bioactivity / 中华骨科杂志

Objective:To explore the effect of decalcified bone matrix (DBM) rich in biological activity on surgical-grade medical calcium sulfate, and to observe the change of different content of DBM on the physical and chemical properties of calcium sulfate, which provide theoretical basis for the preparation of calcium sulfate bone cement with osteogenic and injectable properties.Methods:DBM with weight content of 0, 5%, 10%, 20%, 30%, 40% was fully mixed with CSH. Dissolve 0.3 g of methyl cellulose in 10 ml of deionized water to prepare a 3% methyl cellulose solution. Methylcellulose solution was added according to the liquid-solid ratio of 0.4. The mixture was evenly stirred to form slurry, then the degradation rate, compressive strength, setting time and and pH value of calcium sulfate in vitrowas measured.Results:The initial setting time and final setting time of calcium sulfate were 4.96±0.20 and 5.83±0.12 min respectively. With the increase of DBM content, the initial setting time and final setting time increased significantly ( F=49.275, P<0.05; F=124.859, P<0.05). The compressive strength of pure calcium sulfate is 23.33±6.35 MPa; when the content is 40%, the compressive strength is only 3.33 MPa. With the increase of DBM content, the compressive strength first increased and then decreased; the content of 5%, 10%, 20% DBM had little effect on the compressive strength ( P>0.05), while the compressive strength of 30% and 40% groups decreased significantly ( t=3.259, P<0.05). DBM with different contents can significantly change the degradation rate of calcium sulfate complex. When the content of DBM is 30% and 40%, the complete degradation time in vivo is only 10 d, while the degradation rate of calcium sulfate is 63% in 30 d. At any time point in vitro degradation, DBM had no significant effect on the pH value of calcium sulfate complex culture medium, and the change law was consistent with that of pure calcium sulfate. Conclusion:With the increase of DBM content, the degradation rate is gradually accelerated, the compressive strength is reduced, and the setting time is prolonged, which is not conducive to the preparation of injectable calcium sulfate cement.

Comparative study of three different decalcifying solutions in bone metastasis specimens with breast cancer / 中华病理学杂志

Objective@#To investigate the optimal strategy for immunohistochemical (IHC) staining in bone metastasis specimens from breast cancer.@*Methods@#Twenty-eight bone metastases specimens from breast cancers were divided into three groups and subjected to different decalcifying agents (group A-10% nitrate, group B-EDTA decalcification, and group C-imported decalcifying solution RapidCal). The effects of those on HE and IHC staining for Ki-67, ER, PR, GATA3, RANK, RANKL, HER2 and HER2 FISH results were assessed.@*Results@#There were no significant differences among three groups in HE morphology and IHC staining. Antigen content in the RapidCal group were all intact; the EDTA group showed a similar staining rate, which was better than the nitrate group (P<0.05). Nitrate group showed marked reduction in nuclear Ki-67 staining, but the loss of cytoplasmic antigens (RANK, RANKL) was less than cell membrane antigen (HER2). For FISH, the RapidCal group and EDTA group showed same results, concordant with IHC staining results. The expression of HER2 protein in the nitric acid group was significantly decreased and chromosome 17 labelling was lost (P<0.05).@*Conclusions@#RapidCal treated bone metastases specimens from breast cancer show excellent sample quality in morphological, IHC and FISH results compared with traditional decalcifying agents. Owing to the longer time of EDTA decalcification, the new decalcifying agent RapidCal plays an important role in quality control and clinical application.

An Experimental Study for Minimum Level of Decalcification to Detect the Osteolytic Bone Metastasis of Long Bone on Plain Radiography

BACKGROUND: In 1951, Ardran reported that metastatic bone lesions could be detectable on plain radiography with 30% to 50% of decalcification. Authors performed experimental study for minimum level of decalcification to detect the osteolytic bone metastasis of long bone with recent technique of radiographs. METHODS: One pair of fibula and humerus from two cadavers was cut into specimen 1 inch in length. Distal half of specimen was dipped into hydrochloride (HCl) with 15 min interval. All 16 specimens were checked by film-type radiography (FR), computed radiography (CR), digital radiography (DR). To exclude inter-observer's variance, 3 radiologists evaluated images. Calcium amount before and after decalcification was measured and expressed in percentage of decalcification. RESULTS: Osteolytic changes were detectable with 11% to 16% of decalcification for fibula and 3% to 8% for humerus on plain radiography with FR, CR, and DR. CONCLUSIONS: Our study showed that minimum of 3% and maximum of 16% of decalcification is necessary when osteolytic metastatic bone lesions of long bone could be detected on plain radiography.

Proposal of an Appropriate Decalcification Method of Bone Marrow Biopsy Specimens in the Era of Expanding Genetic Molecular Study

BACKGROUND: The conventional method for decalcification of bone specimens uses hydrochloric acid (HCl) and is notorious for damaging cellular RNA, DNA, and proteins, thus complicating molecular and immunohistochemical analyses. A method that can effectively decalcify while preserving genetic material is necessary. METHODS: Pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl. Pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the EDTA versus HCl comparison group, and 25 cases to the RDO versus HCl comparison group. The decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. For molecular analysis, we randomly selected 5 cases from the EDTA versus HCl and RDO versus HCl groups. RESULTS: The decalcification time for appropriate histomorphologic analysis was the longest in the EDTA method and the shortest in the RDO method. EDTA was superior to RDO or HCl in DNA yield and integrity, assessed via DNA extraction, polymerase chain reaction, and silver in situ hybridization using DNA probes. The EDTA method maintained intact nuclear protein staining on immunohistochemistry, while the HCl method produced poor quality images. Staining after the RDO method had equivocal results. RNA in situ hybridization using kappa and lambda RNA probes measured RNA integrity; the EDTA and RDO method had the best quality, followed by HCl. CONCLUSIONS: The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.

Comparison of micro-computerized tomography and cone-beam computerized tomography in the detection of accessory canals in primary molars

PURPOSE: This study was performed to compare the accuracy of micro-computed tomography (CT) and cone-beam computed tomography (CBCT) in detecting accessory canals in primary molars. MATERIALS AND METHODS: Forty-one extracted human primary first and second molars were embedded in wax blocks and scanned using micro-CT and CBCT. After the images were taken, the samples were processed using a clearing technique and examined under a stereomicroscope in order to establish the gold standard for this study. The specimens were classified into three groups: maxillary molars, mandibular molars with three canals, and mandibular molars with four canals. Differences between the gold standard and the observations made using the imaging methods were calculated using Spearman's rho correlation coefficient test. RESULTS: The presence of accessory canals in micro-CT images of maxillary and mandibular root canals showed a statistically significant correlation with the stereomicroscopic images used as a gold standard. No statistically significant correlation was found between the CBCT findings and the stereomicroscopic images. CONCLUSION: Although micro-CT is not suitable for clinical use, it provides more detailed information about minor anatomical structures. However, CBCT is convenient for clinical use but may not be capable of adequately analyzing the internal anatomy of primary teeth.

Estudo comparativo da morfologia da polpa dentária de dentes humanos submetidos à técnica de clivagem e a técnicas de descalcificação química / Comparative study of dental pulp morphology from human teeth submitted to cutting technique or chemistry decalcification technique

Os rápidos avanços do conhecimento acerca do reparo e regeneração tecidual, têm despertado o interesse pela biologia pulpar. Entretanto, para avaliar microscopicamente a dinâmica do tecido pulpar, é necessário, inicialmente, que o dente seja submetido aos processamentos histológicos de fixação e descalcificação. A descalcificação pode afetar o grau de coloração e pode causar desnaturação de proteínas. Além disso, é um processo demorado, visto que o dente requer um longo período de desmineralização. Assim, a proposta deste trabalho foi de avaliar qualitativamente a matriz extracelular e as células da polpa dentária, comparando três grupos: dois em que o tecido dentário foi descalcificado e um em que a polpa foi removida dos tecidos duros, não necessitando do processo de descalcificação. Dez pré-molares foram fixados em formalina tamponada a 10% por 24 horas. Após, estes dentes foram divididos em três grupos: 4 dentes foram submetidos a processo de descalcificação por meio de solução de Morse, 3 por meio de solução de EDTA a 10% e; os 3 dentes restantes tiveram sua polpa separada dos tecidos duros dentários por meio da técnica de clivagem. Na seqüência, os três grupos foram processados por meio da técnica histológica de rotina e foram corados com H/E. Os resultados desta análise demonstraram que houve uma melhor conservação tanto da matriz extracelular, quanto das estruturas celulares no grupo da clivagem, seguido do grupo Morse e por fim, com a menor conservação das estruturas pelo grupo EDTA.

The rapid advances of the knowledge of repair and regeneration tissues had proved to be an exciting time for pulp biology. However, to study the dynamic of pulp tissue, it is necessary, initially, that the tooth be submitted to histological fixation and decalcification processing. Decalcification may affect the degree of staining and it may cause denaturation of proteins. Furthermore, it is a slow process, demanding long demineralization times for a tooth. Thus, the purpose of the present study was to compare, qualitatively, the pulp extracellular matrix and the pulp cells, submitted to different techniques: EDTA solution decalcification, Anna Morse solution decalcification and a last group which pulp was removed from tooth without decalcification. Ten premolar teeth were fixed in 10% buffered formalin for 24 hours. After this, the teeth were divided in three groups: 4 teeth underwent decalcification with Morse solution; 3, decalcification with 10% EDTA solution and; 3, were sectioned and their pulps were gently removed. Subsequently, the groups followed the routine histological technique and staining with H/E. The results demonstrated that both conservation of pulp cells and extracellular matrix were better in the group without decalcification, followed by the Morse group and, the last, with the worst structures conservation for the EDTA group.

Long-term clinical observation on treatment of delayed union and nonunion of long bone fractures by using decalcification bone matrix / 中华创伤杂志

Objective To explore remote effect of decalcification bone matrix (DBM) in treatment of delayed union and nonunion of long bone fractures. Methods From 1986, 96 cases of delayed union and nonunion of long bone fractures were treated with DBM. Of all, 38 cases of nonunion of long bone were treated with surgical implantation, 37 delayed union and 21 nonunion with percutaneous injection. Results All 96 cases were followed up for 4-16 years (average 7.5 years). Of 37 cases of delayed union treated with percutaneous injection, 35 attained bone union but 2 resulted in nonunion of tibial fractures with a union rate of 94.6%. Of 21 cases of bone nonunion treated with percutaneous injection, 17 got bone union but 4 did nonunion (3 tibial nonunion and 1 humeral nonunion) with a union rate of 80.1% and union time for 3-8 months (average 4.5 months). Of 38 cases treated with implantation of DBM, 36 had bone union but 2 nonunion at 1/3 inferior to the tibia with a union rate of 94.7%. Conclusions Either percutaneous injection or surgical implantation of DBM can attain satisfactory effect even to autograft. After prepared by dehydration, degrease, decalcification and irradiation disinfection, DBM is characterized by safer transplantation and less immunity than other bone transplantations and can be preserved for a long time so as to be applied both in the peacetime and the wartime.