Relationship between pre-radiographic cartilage damage following anterior cruciate ligament injury and biomarkers of cartilage turnover in clinical practice: a cross-sectional observational study.

OBJECTIVE: To determine whether differences in synovial fluid (SF) biomarkers of collagen and proteoglycan turnover are associated with pre-radiographic damage to articular cartilage and menisci following anterior cruciate ligament (ACL) injury and are of clinical value. METHOD: SF samples from ACL injured knees of 108 patients were obtained when damage to cartilages and menisci was evaluated arthroscopically. Concentrations of SF collagenase-generated cleavage neoepitope of type II collagen (C2C) were determined using ELISA and aggrecan-derived disaccharides of chondroitin-4-sulfate (Δdi-C4S), chondroitin-6-sulfate (Δdi-C6S), and keratan sulfate (KS), were measured in SF by High performance liquid chromatography (HPLC). RESULTS: Radiographic examination failed to detect any intra-articular degenerative changes. The number of high-grade cartilage lesions was positively associated with age, duration after injury and the level of C2C, and negatively with the level of KS. There was no association between the number of high-grade cartilage and meniscal lesions. Multivariable logistic regression revealed significant associations of increased C2C (adjusted Odds ratio (OR) of the upper quartile to remainder of 2.49, 95% Confidence interval (CI) = 0.85-7.27) and decreased KS (adjusted OR of the lower quartile to the remainder of 3.32, 95% CI = 1.19-9.24) with the presence of three or more high-grade cartilage lesions, independent of age and duration after injury. The combined impact of increased C2C and decreased KS was 22.8 (95% CI = 1.95-265.9), far exceeding the impact of each independent biomarker. CONCLUSION: Combinations of the C2C and KS as described here may offer greater ability to identify patients with early pre-radiographic high-grade cartilage damage compared to single clinical or biomarker parameters.

Peroxidase activity in the submandibular gland of the house musk shrew, Suncus murinus (Soricidae, Insectivora).

Endogenous peroxidase activity in the submandibular gland of the house musk shrew, Suncus murinus was cytochemically investigated by light and electron microscopy using 3,3'-diaminobenzidine-tetrahydrochloride salt (DAB). The submandibular glands of male Suncus murinus at 8-month-olds were excised and diced into small pieces. In general, salivary glands are structurally divided into a terminal portion comprising a secretory portion and duct system. The submandibular gland of the Suncus murinus, the terminal portions consisted of proximal and distal acinar cells. On the other hand, a granular duct cell of the duct system contained a number of characteristic myelin-like bodies. In the present study, the peroxidase reaction products were localized in the secretory granules of the proximal acinar cells and in the endoplasmic reticulum, Golgi apparatus and myelin-like bodies of the granular duct cells. These reaction products were reduced when 5 mM 3-amino-1,2,4-triazole was added to the reaction medium. Additionally, release of peroxidase into the lumen was observed. In conclusion, the proximal acinar and granular duct cells formed peroxidase and may have performed excretory secretions. Moreover, the peroxidase positive myelin-like body consisted of lamellated membrane and its outer surface membrane continued to the endoplasmic reticulum.

An odontometrical difference in the mandibular molars of two laboratory strains of the musk shrew, Suncus murinus, derived from Bangladesh and Tokunoshima Island of Japan.

We investigated an odontometrical difference in the mandibular molars (M1, M2, and M3) of two laboratory strains of the musk shrew (Suncus murinus) originating in Bangladesh (BAN strain) and Tokunoshima Island of Japan (TKU strain). We used skulls from two strains of shrews that were maintained under identical laboratory conditions. Mesiodistal and buccolingual crown diameters in the trigonid and talonid of the mandibular molars were measured with a measuring microscope, calibrated to 0.001 mm. The crown proportion was expressed by the crown indices calculated from the measurements. Size reduction was analyzed quantitatively according to the reduction index. All crown dimensions were significantly larger in BAN shrews than in TKU shrews (P < 0.01). Sexual differences were noted in the talonid dimensions, while interstrain differences were clearly evident in the trigonid dimensions. The crown indices in M1 showed the least interstrain difference of the three molars. The crown indices showed that TKU shrews had relatively larger buccolingual diameters and talonid diameters than BAN shrews, and the reduction indices showed that TKU shrews had relatively larger M2 and M3 than BAN shrews. To extract the variance components of tooth shape, a principal component analysis was performed after the variables were standardized. After Varimax rotation, each factor was interpreted. The first three factors accounted for 79.9% of all variances. The first component represented the mesiodistal crown proportion of the trigonid-to-talonid crown component. The second and third components represented the relative size of buccolingual diameters in the distal molars for M1. The principal component scores showed that TKU shrews had relatively larger talonids and distal molars than BAN shrews.

Crown structure of the maxillary molars in the Japanese shrew mole, Urotrichus talpoides (Insectivora, Talpidae).

The maxillary first and second molars (M1 and M2) in the Japanese shrew mole, Urotrichus talpoides, were investigated using an odontometrical approach. The mesiodistal crown diameter was larger in M1 than in M2, while the buccolingual diameter of M1 was nearly equal to that of M2. M2 was more compressed mesiodistally than M1. M1 had a large distal triangle on the stylar shelf. The mesial triangle of M2 was slightly larger than the distal triangle. Despite being smaller than M1, M2 was less variable than M1 in terms of size. The distal triangle of M1 and the mesial triangle of M2 were well developed, and thus this area, which corresponds to the inflection point of the maxillary dental arch, was most likely the center of an occlusal function.

Crown dimensions of the maxillary molars in Tupaia glis.

The crown dimensions of the maxillary molars in Tupaia glis were measured, and the most common molar size sequence was M1 > M2 > M3. The M2 and M3 molars were smaller than the M1 in the mesiodistal crown diameters. With regard to the buccolingual diameters, the distal part of M1 and mesial part of M2 were relatively larger and less variable in size. This stable area corresponded to the inflection point of the maxillary arch curve. These results could be explained from a functional morphological standpoint.

A morphological study on the crown dimensions of the mandibular molars in Tupaia glis.

The crown dimensions of the mandibular molars in Tupaia glis were measured. All the mean values of crown diameters and areas were larger for the M1 molar than for the M2 and M3. The last two molars were more reduced in the talonid component than in the trigonid compared to the M1, and were more reduced in the buccolingual than in the mesiodistal direction. The most common molar size sequence (MSS) was M1 > M2 > M3, and this pattern was more frequently observed in the talonid component than in the trigonid. In the canonical discriminant analysis, all the cases of the M3 were discriminated correctly, but some cases of the first two molars were confused with each other. The molars size of the mandible was closely related to that of the maxilla.

The effect of supragingival plaque control on the subgingival microbiota in subjects with periodontal disease.

The present investigation was performed to study the effect on the subgingival microbiota, of a plaque control program which included meticulous oral hygiene instruction, supragingival scaling and professional monitoring during a 2 year period. 300 subjects were examined for periodontal disease and monitored for 2 years without treatment. After the 2 year examination, 80 subjects were invited to participate in a treatment program intended to improve the standard of their self-performed plaque control. 40 of the invitees had a gingivitis and only minor attachment loss, while 40 subjects had moderate signs of periodontitis. 62 subjects volunteered for this treatment. 23 of the volunteers (Group AB) had several sites with deep pockets (> 4 mm). 39 of the volunteers had gingivitis but shallow pockets only (Group C). Group AB contributed 31 shallow pocket sites (A-sites) and 40 deep pocket sites (B-sites), while Group C contributed 63 shallow sites (C-sites). After the clinical examination, samples of the subgingival microbiota were harvested from the 134 A, B and C sites. The 62 subjects were enrolled in a supervised oral hygiene program. Supragingival scaling was carried out. Oral hygiene instruction was provided and repeated on an individual need basis so that all subjects reached and maintained a supragingival plaque score which was < 20%. 24 months after the year 2 examination, the 62 subjects were examined again using both clinical and microbiological examination procedures. The findings demonstrated that carefully performed supragingival plaque control changed the quantity and the composition of the supragingival microbiota.(ABSTRACT TRUNCATED AT 250 WORDS)

Mandibular dysfunction and periodontitis. A comparative study of patients with periodontal disease and occlusal parafunctions.

Fifty-one patients (mean age, 47.3 years) with moderate to severe periodontal disease and 40 patients (mean age, 48.9 years) with symptoms related to bruxism (occlusal parafunctions such as grinding and/or clenching of the teeth) were compared with regard to periodontal conditions and signs and symptoms of mandibular dysfunction. The bruxists reported more symptoms of pain and dysfunction of the masticatory system than the periodontal patients. The clinical dysfunction index was significantly higher among the bruxists, while there was a similarity between the groups in the variation of occlusal conditions, except for occlusal wear, which was more pronounced in the bruxist group. Attrition was in general positively correlated to alveolar bone height. This correlation was stronger (and statistically significant) for the canines than for other teeth. Attrition was negatively correlated to tooth mobility. It is concluded that patients with moderate to severe periodontal disease and patients with bruxism/occlusal parafunctions are distinctly different with regard to signs and symptoms of mandibular dysfunction. The results support the opinions that there is no or only weak correlation between periodontal disease and bruxism, and between bruxism and occlusal status.

Periodontal status and bruxism. A comparative study of patients with periodontal disease and occlusal parafunctions.

This study investigated a possible association between bruxism and severity of periodontal disease. Subjects consisted of 51 patients (mean age 47.3 years) referred to the Department of Periodontology for treatment of moderate to severe periodontal disease (Perio-group) and 40 patients (mean age 48.9 years) referred to the Department of Stomatognathic Physiology for treatment of symptoms related to bruxism (Bruxism-group). Examination of the two groups included measurements of the alveolar bone height, probing attachment level, tooth mobility, and attrition of teeth. A questionnaire was also used to gain information on the patient's awareness of bruxism and tooth mobility. Awareness of clenching and/or grinding was reported by 57% of patients in the Bruxism-group and 24% of patients in the Perio-group. The perio-patients reported significantly higher frequency of tooth mobility than did the bruxism-patients. Alveolar bone loss, attachment loss, and tooth mobility were significantly more pronounced in the Perio-group than in the Bruxism-group. The Bruxism-group showed a higher frequency of tooth attrition than the Perio-group. Periodontal disease and bruxism seldom occurred in the same individual, and the results indicate that the two phenomena are in general not closely associated.

Nature of bone morphogenetic protein (BMP) from decalcified rabbit bone matrix.

Rabbit bone morphogenetic protein (BMP) from demineralized and defatted rabbit bone matrix was partially purified. BMP activity was examined by the implantation of fractionated materials into the thigh muscle pouch of the mouse. Rabbit BMP was solubilized by both 4M guanidine hydrochloride (GuHCl) and 6M urea solutions. Crude BMP had isoelectric point precipitation at pH 3 in 6M urea and showed bone morphogenesis. Fractions eluted with 0 and 0.2 N NaCl in DEAE CL-6B ion exchange chromatography showed bone morphogenesis in each individual pH of pH 4 to pH 7 but the fraction eluted with 1.0 N NaCl did not show any activity. Sephadex G-75 filtration separated the crude material into three peaks and the peak of about 23,000 showed bone morphogenesis. In sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and isoelectric focusing, rabbit BMP was thought to be an acidic protein having a molecular weight of 24,000 with an isoelectric point around 4.85.

Chemosterilized autolyzed antigen-extracted allogeneic (AAA) bone matrix gelatin for repair of defects from excision of benign bone tumors: a preliminary report.

In a series of 33 bone graft operations, antigen-extracted, autolyzed, allogeneic (AAA) bone matrix gelatin was substituted for autologous bone. The period of follow-up was 2.0 to 3.5 years. AAA bone gelatin was resorbed more rapidly than whole bone. AAA bone gelatin was replaced by new bone in the same intervals of time as observed with autologous bone. In treatment of bone tumors with AA bone gelatin, the results of the operation depend upon the nature of the pathologic processes in the host bed. In normal host bed, tight contact between implant and recipient bone is essential for success. The overall results of a preliminary study of 33 cases of 91% successful.

Solubilization and purification of bone morphogenetic protein (BMP) from Dunn osteosarcoma.

The 4 M GuHCl soluble proteins of Dunn osteosarcoma were fractionated in two steps by means of CsCl density gradient centrifugation. Further purification of bone morphogenetic protein (BMP) was accomplished by molecular sieve techniques utilizing thin-channel diafiltration (Amicon), Sepharose CL-6B and Sephacryl S-200 gel filtration. Partially purified BMP fractions were analyzed by electrophoresis on 7.5% SDS polyacrylamide gel. The molecular weight of BMP active fractions ranges from 30,000 to a few thousand daltons. BMP may be one of the two proteins of MW of 12,500 and 16,000 daltons.

Solubilized bone morphogenetic protein (BMP) from mouse osteosarcoma and rat demineralized bone matrix.

A selection of proteins including bone morphogenetic protein (BMP) was extracted in a disaggregated form from Dunn osteosarcoma or rat demineralized bone matrix by 4M guanidine hydrochloride (GuHCl) solution without losing its biological activity. The GuHCl extracts of Dunn osteosarcoma were divied into 4 different fractions by cesium chloride (CsCl) density gradients. Under a dissociative condition, the highest new bone yield was obtained in the low dense top one-third fraction, and BMP acitivity declined with increase in the density of each fraction. No BMP potential was observed in the surface-gel fraction under dissociative conditions. Under an associative condition (low GuHCl concentrations), BMP activity appears in the surface-gel fraction, while under a dissociative condition (high concentrations of GuHCl) BMP appears in the fraction below the surface gel. These facts suggest that under associative conditions, BMP aggregates with other low dense proteins in the surface-gel fraction and that this may be the state of aggregation of BMP in cells and matrix in nature. Present observations support the assumption that BMP is a relatively low density protein and excludes the idea of BMP activity in the collagen molecule, per se. A specific protein, with an apparent molecular weight of 63,000 daltons, is present in all fractions that exhibit BMP activity, and absent in fractions that do not exhibit this activity. BMP is not species-specific; rat BMP induces bone formation in mice. CsCl density-gradient centrifugation is an efficient tool for further purification and isolation of BMP.

Osteogenesis and chondrogenesis in transplants of Dunn and Ridgway osteosarcoma cell cultures.

Dunn osteosarcomas synthesize 2 times more alkaline phosphatase than do Ridgeway osteosarcomas, 3 times more than do HeLa cells, and 4 to 5 times more than do rat or mouse fibroblast cell cultures. Implants of killed freeze-dried Dunn cell cultures into the thigh muscles are resorbed and replaced by normal cartilage, bone, and bone marrow tissue, while implants of freeze-dried Ridgeway cells are resorbed and replaced by fibrous tissue only. Outgrowths of normal muscle septum connective tissue cells onto the stroma of Ridgeway tumors differentiate into fibrous tissue. Cultures of either tumor on a substratum of bone matrix stroma prepared from normal bone proliferate, assume a spherical shape, and perpetuate the transformed osteoblast-like cell without forming attachments or adapting to the contour of the substratum. Outgrwoths of muscle mesenchymal cells on the Dunn tumor stroma differentiate into cartilage. Dunn osteosarcoma cell cultures proliferate on the inside and produce deposits of normal bone (not tumorous bone) on the outside of diffusion chambers. Killed freeze-dried cell cultures produce transfilter deposits of normal bone and bone marrow, but the quantity is significantly lower. On a substratum of cellulose acetate, outgrowths of muscle connective tissue will differentiate into cartilage when cell-free Dunn stroma is present under the organ culture grid. Tumorigenesis and normal cartilage and bone morphodifferentiation are antithetic, but tumor cells transfer a bone morphogen similar to the bone morphogenetic protein (BMP) of normal bone matrix. BMP recruits mesenchymal cells to proliferate and differentiate into cartilage and bone.

An osteosarcoma cell and matrix retained morphogen for normal bone formation.

Histophysiology, ultrastructure, chemical analyses of transplants and implants of Dunn and Ridgway mouse osteosarcomas demonstrate that tumorigenesis is a manifestation of deranged morphogenesis in developing mesenchymal cell populations. The end product of development is defective, incompletely calcified, disorganized bone without any inclusions of bone marrow tissue. When Dunn osteosarcoma is freeze-dried and then implanted, the tumor is resorbed and replaced by deposits of normal cartilage, bone, and bone marrow. Freeze-dried Ridgway osteosarcoma is replaced only by a fibrous connective tissue scar. Disaggregated Dunn tumor osteoblasts synthesize a trypsin-labile collagenase-resistant cell surface localized bone morphogen. Tumor matrix stroma, prepared by sequential chemical extraction of soluble non-collagenous proteins also contains significant quantities of the same bone morphogen. Tumor tissue pulverized to particle size as small as 44 micrometer3 transmitted bone morphogen more rapidly than intact tumor tissue. The total tumor cell and stroma mediated bone morphogen produces three times more normal bone than normal cortical bone matrix. Our working hypothesis is that a normal bone morphogenetic polypeptide (BMP) is synthesized by Dunn osteosarcoma cells and retained by the tumor matrix stroma. Neither the mechanism of transmission nor the mesenchymal cell receptor sites of BMP are known.