Large Numbers Of Osteoclasts Research Articles

Патогенез и морфогенез хронического асептического остеомиелита

Chronic nonbacterial (recurrent) osteomyelitis is a rare inflammatory disease of the musculoskeletal system with unknown etiology described in 1972 by A. Gidion et al. as subacute or chronic symmetrical osteomyelitis. This article reviews literature on pathogenesis and morphology of the disease and studies balance violation of pro- and anti-inflammatory cytokines and its association with osteolysis. Morphological manifestations in bone biopsies are given. We describe necrosis and thinning of trabeculae in combination with sclerosis of intertrabecular spaces and pronounced lymphohistiocytic infiltration with a large number of osteoclasts. Due to the absence of specific signs, the necessity of clinical and morphological correlations and genetic research in differential diagnosis is emphasized. Keywords: chronic nonbacterial osteomyelitis, chronic recurrent osteomyelitis, autoinflammatory disease

LRP1-deficient leptin receptor-positive cells in periodontal ligament tissue reduce alveolar bone mass by inhibiting bone formation

ObjectiveLeptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells. DesignBone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers. ResultsBone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly. ConclusionLRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue.

Absence of Tumor Necrosis Factor Receptor 1 Inhibits Osteoclast Activity in Apical Dental Resorption Caused by Endodontic Infection in Mice

IntroductionThe aim of this study was to evaluate osteoclastogenesis and dental resorption resulting from endodontic infection in wild-type (WT) and tumor necrosis factor receptor 1 genetically deficient (TNFR1 KO) mice. MethodsAfter approval by the ethics committee on the use of animals, 40 mice were distributed into 2 experimental groups based on time periods: 14 days (n = 10 WT mice and n = 10 TNFR1 KO mice) and 42 days (n = 10 WT mice and n = 10 TNFR1 KO mice). After these periods, morphometric analysis was performed using bright field and fluorescence microscopy and tartrate-resistant acid phosphatase histoenzymology to identify osteoclasts. One-way analysis of variance followed by the Tukey post hoc test was used for the statistical analysis (α = 0.05). ResultsWT mice in the 42-day period had a greater apical dental resorption in the distal root of the first molar than TNFR1 KO mice (P < .05). On the other hand, TNFR1 KO mice showed a smaller number of osteoclasts on the dental surface than WT mice (P < .05). ConclusionsWT mice with apical periodontitis had more extensive apical dental resorptions and a larger number of osteoclasts on the tooth surface than TNFR1 KO mice.

Tumour necrosis factor-α promotes BMHSC differentiation by increasing P2X7 receptor in oestrogen-deficient osteoporosis.

The exact mechanism of tumour necrosis factor α (TNF‐α) promoting osteoclast differentiation is not completely clear. A variety of P2 purine receptor subtypes have been confirmed to be widely involved in bone metabolism. Thus, the purpose of this study was to explore whether P2 receptor is involved in the differentiation of osteoclasts. Mouse bone marrow haematopoietic stem cells (BMHSCs) were co‐cultured with TNF‐α to explore the effect of TNF‐α on osteoclast differentiation and bone resorption capacity in vitro, and changes in the P2 receptor were detected at the same time. The P2 receptor was silenced and overexpressed to explore the effect on differentiation of BMHSCs into osteoclasts. In an in vivo experiment, the animal model of PMOP was established in ovariectomized mice, and anti‐TNF‐α intervention was used to detect the ability of BMHCs to differentiate into osteoclasts as well as the expression of the P2 receptor. It was confirmed in vitro that TNF‐α at a concentration of 20 ng/mL up‐regulated the P2X7 receptor of BMHSCs through the PI3k/Akt signalling pathway, promoted BMHSCs to differentiate into a large number of osteoclasts and enhanced bone resorption. In vivo experiments showed that more P2X7 receptor positive osteoclasts were produced in postmenopausal osteoporotic mice. Anti‐TNF‐α could significantly delay the progression of PMOP by inhibiting the production of osteoclasts. Overall, our results revealed a novel function of the P2X7 receptor and suggested that suppressing the P2X7 receptor may be an effective strategy to delay bone formation in oestrogen deficiency‐induced osteoporosis.

REMODELING OF THE TEMPROMANDIBULAR JOINT FOLLOWING DIFFERENT RATES OF MANDIBULAR DISTRACTION OSTEOGENESIS (AN EXPERIMENTAL STUDY)

Introduction: patients with craniofacial deformities resulting from trauma, infection, tumors and congenital defects usually require skeletal expansion. Tradition treatment methods, involve autogenous bone grafting, which often results in problems including graft resorption, infection and donor site morbidity. Distraction osteogenesis has been adopted more and can help overcome these problems. Aim of the work: the purpose of this study was to compare histologically the effect of bilateral distraction osteogenesis of the mandibular body at different rates on the integrity of the condylar cartilage in rabbits. Material and Methods: This study was performed on 2 groups of twelve healthy rabbits (adult male) : (group 1 and group 2) each group (n=6 rabbits, and 12 joints). Two rabbits (4 joints) in each subgroup according to the post distraction period (1, 2, and 4 weeks). In group 1, the distraction was performed daily at rate of 1 mm for 5 days. In group 2, the distraction was performed twice daily at rate of 1.5 mm (3 mm per day) for 5 days.Results: Animals in both groups (1 and 2) showed changes in condylar head, which seemed to be reversible according to the post distraction period. After the first week, condylar layers were decreased in thickness with irregular fibrous outline, bone trabeculae were decreased in number and invaded by large number of osteoclasts. After the second week, the condylar head showed evidence of regeneration of all layers including the disc. After the fourth week, the subchondral area started to be refilled with new bone with numerous osteoblast. Group 2 (3 mm per day) showed more bone and collagen formation.Conclusion: this experimental study reported that distraction rates of 1 mm per day and 3 mm per day showed degenerative change in the TMJ condylar cartilage in the first post distraction week. At the second week, all the condyles showed adaptive and remodeling signs. Regeneration to the normal histological structure appeared in the fourth post distraction weeks, in group 2 (3 mm per day) more than in group 1 (1 mm per day).

Efficacy of Photodynamic Therapy in Minimizing Bisphosphonate-Related Osteonecrosis of the Jaws After Dental Extraction: A Preliminary Animal Study

Except for a few case reports, there is no study on the efficacy of photodynamic therapy (PDT) in decreasing or preventing bisphosphonate-related osteonecrosis of the jaws (BRONJ). This preliminary animal study assessed the effectiveness of this clinical treatment. Zoledronic acid was administered to 20 rats for 5weeks. Two weeks later, a first molar was extracted from each rat. The rats were randomized to control and PDT groups. PDT was performed in the experimental group after surgery and at weeks 1, 2, 3, 4, 5, 6, and 7 after surgery. In the 8th week, BRONJ signs were evaluated by an observer blinded to randomization. Rats were euthanized and underwent histopathologic and histomorphometric evaluations. Clinical signs were compared using the Fisher test. Histomorphometric parameters were compared using the Mann-Whitney U test (α=0.05). Two rats were lost from each group. Bone exposure decreased from 7 rats in the control group to 1 rat in the PDT group (P=.010). The stage of BRONJ decreased significantly from 7 rats in stage 1 to only 1 rat in stage 1 (P=.010). PDT decreased inflammation considerably for gingival eosinophils and lymphocytes and bone neutrophils, eosinophils, and lymphocytes. A larger percentage of live bone and smaller percentages of necrotic bone, empty lacunae, and neovascularization were observed in the PDT group. PDT also maintained bone remodeling, indicated by a large number of osteoclasts (P≤.001 for all comparisons by Mann-Whitney U test). Within the limitation of this preliminary animal study, PDT was found to be considerably effective clinically and histopathologically in decreasing or preventing BRONJ in rats. Future human studies are needed to verify these results.

Macroscopic and microscopic evaluation of flapless alveolar perforations on experimental tooth movement.

Objective: The aim of this study was to evaluate a flapless surgical technique as an alternative to traditional alveolar corticotomy used to accelerate orthodontic tooth movement (OTM). Methods: To induce OTM in Wistar rats, 40 cN of orthodontic force were applied to the maxillary left first molars. Forty rats were distributed into control groups (CG1, CG3, CG7 and CG14) and experimental groups (n= 5), in which alveolar perforations were made using a spear-shaped guide bur (EG1, EG3, EG7, EG14). Euthanasia dates were set at 1, 3, 7 and 14 days, respectively, after tooth movement began. The amount of OTM was measured with a caliper, and osteoclasts present in the periodontal ligament of the mesial root of the moved tooth were counted by means of histological evaluation (tartrate-resistant acid phosphatase staining, TRAP). Results: Although there was no difference in the amount of OTM within subgroups of corresponding experimental periods (p> 0.05), when EG14 and CG14 were compared, a larger number of osteoclasts was counted in the experimental group (p< 0.00). Conclusion: The authors concluded that flapless cortical alveolar perforations led to more intense osteoclastic activity on the fourteenth day; nevertheless, no evidence of accelerated OTM could be noted.

Chemical characterization of wound ointment (WO) and its effects on fracture repair: a rabbit model

BackgroundWound ointment (WO), a kind of Chinese medicine, can significantly promote fracture healing. The study aimed at analyzing the chemical composition and the effects of WO on fracture of rabbits and tried to explore the corresponding molecular mechanism in cytokine.MethodsThe qualitative and quantitative analysis of WO was conducted by liquid chromatography-mass spectrometry (LC–MS). Fifty-four Zealand mature male rabbits were randomly divided into 3 groups: Control group, Yunnan Baiyao (YB) group and WO group. All the rabbits suffered a fracture of right radius and were then stabilized with an external fixator. Treated with different methods, fracture healing was observed. The bone specimens were subjected to radiograph, immunohistochemistry (IHC) analysis, hematoxylin–eosin staining (HE), western blot and enzyme linked immunosorbent assay (ELISA).ResultsA total of 12 active compositions were detected by LC–MS. Radiographs showed a considerably better bone healing and remodeling of the fracture in WO group. HE experiments showed that a large number of osteoclasts appeared in the early stage when treated with WO. In immunohistochemistry (IHC), western blot and ELISA test, significant increases in vascular endothelial growth factor (VEGF) expression were observed in WO group compared with other two groups.ConclusionsWound ointment contained active compositions which efficiently promoted fracture healing through increasing the expression of VEGF.Trial Registration Not applicable

Bone marrow transplantation improves autoinflammation and inflammatory bone loss in SH3BP2 knock-in cherubism mice

Cherubism (OMIM#118400) is a genetic disorder in children characterized by excessive jawbone destruction with proliferation of fibro-osseous lesions containing a large number of osteoclasts. Mutations in the SH3-domain binding protein 2 (SH3BP2) are responsible for cherubism. Analysis of the knock-in (KI) mouse model of cherubism showed that homozygous cherubism mice (Sh3bp2KI/KI) spontaneously develop systemic autoinflammation and inflammatory bone loss and that cherubism is a TNF-α-dependent hematopoietic disorder. In this study, we investigated whether bone marrow transplantation (BMT) is effective for the treatment of inflammation and bone loss in Sh3bp2KI/KI mice. Bone marrow (BM) cells from wild-type (Sh3bp2+/+) mice were transplanted to 6-week-old Sh3bp2KI/KI mice with developing inflammation and to 10-week-old Sh3bp2KI/KI mice with established inflammation. Six-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells exhibited improved body weight loss, facial swelling, and survival rate. Inflammatory lesions in the liver and lung as well as bone loss in calvaria and mandibula were ameliorated at 10weeks after BMT compared to Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells. Elevation of serum TNF-α levels was not detected after BMT. BMT was effective for up to 20weeks in 6-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells. BMT also ameliorated the inflammation and bone loss in 10-week-old Sh3bp2KI/KI mice. Thus our study demonstrates that BMT improves the inflammation and bone loss in cherubism mice. BMT may be effective for the treatment of cherubism patients.

Effect of aqueous extracts of Scutellaria baicalensis Georgi and Radix paeoniae Alba on the serum IgG1 and IgG2a of the periodontitis mice

To examine the effect of aqueous extracts of Scutellaria baicalensis Georgi and Radix paeoniae Alba on periodontitis mice and compare the results of the two herbs for the treatment of the periodontitis mice. Sixty-four SPF 12-week-old male Kunming mice were selected and randomly divided into four groups:Control group(C); Experimental periodontitis group (P):the peridontitis models in Kunming mice were prepared by wrapping silk ligature and inoculating with putative periodontopathic bacteria; Scutellaria baicalensis Georgi treatment group (SG): periodontitis was induced by the same method described above, the mice were gavaged with Scutellaria baicalensis Georgi; Radix paeoniae Alba treatment group (RG): periodontitis was induced by the same method described above, the mice were gavaged with Radix paeoniae Alba.Four mice were sacrificed at each time point of the end of 4, 6, 8 and 10 weeks in each group. The histopathological changes of periodontal tissue were observed under microscope with HE staining. The level of serum IgG1 and IgG2a was measured by enzyme-linked immunosorbent assay (ELISA) . A serious inflammatory response, alveolar progressive absorption and a large number of osteoclasts were observed in the experimental periodontitis group.However, in SG and RG, the inflammation of the periodontal tissue was decreased and tissue repair was significant. The level of serum IgG2a in SG (6 week:0.934 ± 0.006, 8 week:0.743 ± 0.009, 10 week: 0.674 ± 0.008) and RG (6 week: 1.023 ± 0.032, 8 week: 0.851 ± 0.032, 10 week:0.790 ± 0.009) was significantly decreased after the mice were gavaged with the two herbs(P < 0.01). The level of serum IgG2a in SG was significantly lower than that of RG (P < 0.01). The level of serum IgG1 in SG (6 week: 0.314 ± 0.006, 8 week: 0.344 ± 0.004, 10 week: 0.367 ± 0.006) and RG (6 week: 0.287 ± 0.005, 8 week: 0.303 ± 0.058, 10 week: 0.336 ± 0.006) were significantly increased (P < 0.01). The level of serum IgG1 in SG was significantly higher than that of RG (P < 0.01). Both the aqueous extracts of Scutellaria baicalensis Georgi and Radix paeoniae Alba showed therapeutic effect on periodontitis in mice.Scutellaria baicalensis Georgi was more effective than Radix paeoniae Alba.

Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts

Calcium-deficient hydroxyapatite (HA) granules with a unique spherical shape were prepared using an applied hydrothermal method. Spherical stoichiometric HA granules were also prepared by normal sintering and both granules were used for implantation into rat tibiae to compare the biological responses to each implant. Twelve and 24 weeks after implantation, the volume of calcium-deficient HA granules was significantly less than that of stoichiometric HA granules, and the biodegradability of calcium-deficient HA granules was confirmed. The larger number of osteoclasts, larger osteoblast surface and larger bone volume in the implanted area of calcium-deficient HA than those of stoichiometric HA suggested that osteoclastic resorption of calcium-deficient HA affected osteogenesis in that area. To analyze the direct contribution of osteoclasts to osteogenesis, C2C12 multipotent myoblastic cells, which have the potential to differentiate into osteoblasts in the presence of bone morphogenetic protein 2, were cultured with supernatants of osteoclasts cultured on calcium-deficient HA, stoichiometric HA, β-tricalcium phosphate disks or plastic dishes, or bone marrow macrophages cultured on plastic dishes. Supernatants of osteoclasts but not bone marrow macrophages stimulated the expression of Runx2 and osteocalcin in C2C12 cells in concert with bone morphogenetic protein 2. The expression of alkaline phosphatase was stimulated with supernatants of osteoclasts cultured on ceramic disks. These results suggested that osteoclasts produced certain soluble factors which stimulated osteoblastic differentiation and they were thought to be associated with the induction of a larger osteoblast surface and bone volume in the animals implanted with calcium-deficient HA granules.

Effect of Bilateral Mandibular Osteodistration on the Condylar Cartilage: An Experimental Study on Rabbits

Although various aspects of bone formation during distraction osteogenesis have been studied extensively, there are only limited experimental data concerning the influence of hyper-physiologic mandibular distraction rate on structural alterations in the temporomandibular joint (TMJ) condylar cartilage. The purpose of this study was to evaluate the effect of bilateral distraction osteogenesis of the mandibular body, at a hyper-physiologic rate and length, on the integrity of the condylar cartilage in rabbits. MATERIALS AND METHODS: Eighteen healthy adult male rabbits weighing 2 to 3 kg were assigned to 1 of 2 groups: the control group (n = 2 rabbits, 4 joints) or the study group (n = 16 rabbits, 32 joints) four rabbits (8 joints) in each subgroup according to the post-distraction period (1,2,3 or 4 weeks). In the control group, rabbits received sham surgery (Osteotomy without distraction) and then left to live for 4 weeks under the same condition of the study group then euthanized using intravenous overdose of pentobarbital sodium. In the study group, an extra oral custom-made distracter was employed to achieve bilateral mandibular hyper physiologic distraction (1.5 mm twice daily for 5 days) distraction. All animals were evaluated clinically and histomorphometrically and results analyzed by MINITABE 13.1 statistical package using ANOVA test. RESULTS: Animals underwent distraction showed obvious changes in condylar surface contour related to length of the follow up period, compared to the control; these changes seemed to be partly reversible. The most pronounced observation was the irregularities and resorption in the anterior part of the condylar cartilage and the subcondylar bone. Moreover, at the first two weeks, the area of resorption was invaded by large number of osteoclasts and chronic inflammatory cells which declined later in the 3rd and 4th weeks and replaced with osteoblastic activities. CONSLUSION: These experimental data showed that distraction rate of 3 mm per day may lead to degenerative or even early arthritic changes in the TMJ condylar cartilage in the 1st and 2nd post-distraction weeks. However, all condyles showed adaptive and remodeling sings in the following 3rd and 4th weeks.

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.

Limited Rescue of Osteoclast-Poor Osteopetrosis After Successful Engraftment by Cord Blood From an Unrelated Donor

We report on a case of osteoclast-poor osteopetrosis who received a hematopoietic stem cell graft and, despite hematological engraftment, showed little signs of response in the skeletal defect. Clinical and laboratory studies supported the concept that the bone microenvironment remained abnormal, thus reducing the clinical response to transplantation. Osteopetrosis is a rare genetic disorder characterized by severely reduced bone resorption resulting from a defect in either osteoclast development (osteoclast-poor osteopetrosis) or activation (osteoclast-rich osteopetrosis). Patients with osteoclast-rich osteopetrosis can be rescued by allogenic hematopoietic stem cell transplantation; however, little information exists concerning the success of transplantation as a treatment for osteoclast-poor osteopetrosis. We report on a child with osteoclast-poor osteopetrosis whose diagnosis was delayed, consequently receiving a cord blood transplant from an unrelated donor at the age of 8 years. Engraftment was deemed successful by peripheral blood genotyping, although >3 years after transplantation there was little rescue of the skeletal defect and anemia, and extramedullary hematopoiesis persisted. Peripheral blood mononuclear cells from the osteopetrosis patient, before and after transplantation, were used to generate osteoclasts in vitro in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Before transplantation few, small mononuclear osteoclasts formed (F-actin ring-positive cells, co-localizing with vitronectin receptor [alphavbeta3 integrin] and TRACP) associated with occasional, small resorption lacunae. Low levels of collagen C-terminal telopeptide (CTx) fragments were released from these cultures as assessed by ELISA (CrossLaps; patient, 12.85 nM; control, 448.6 nM). In contrast, osteoclasts formed in cultures after transplantation formed to a similar degree to control cultures from healthy individuals: large numbers of osteoclasts containing numerous nuclei were present, and approximately 50% of the surface of bone slices was resorbed, associated with intermediate levels of collagen fragment release (116.48 nM). The culture data reflect the histopathology and radiological findings and also support previous studies showing that neither M-CSF nor RANKL rescues osteoclast-poor osteopetrosis. This is the first case reported in which a successful hematopoietic engraftment failed to correct an osteopetrotic skeletal defect, and this finding may be credited to the age at which the child was transplanted.

Polyethylene and titanium particles induce osteolysis by similar, lymphocyte-independent, mechanisms

Periprosthetic osteolysis is a major clinical problem that limits the long-term survival of total joint arthroplasties. Osteolysis is induced by implant-derived wear particles, primarily from the polyethylene bearing surfaces. This study examined two hypotheses. First, that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles. Second, that lymphocytes do not play a major role in particle-induced osteolysis. To test these hypotheses, we used the murine calvarial model that we have previously used to examine titanium-induced osteolysis. Polyethylene particles rapidly induced osteolysis in the murine calvaria 5–7 days after implantation. The polyethylene-induced osteolysis was associated with large numbers of osteoclasts as well as the formation of a thick periosteal fibrous tissue layer with numerous macrophages containing phagocytosed polyethylene particles. Polyethylene-induced osteolysis was rapidly repaired and was undetectable by day 21 after implantation. Lymphocytes were noted in the fibrous layer of wild-type mice. However, the amount of osteolysis and cytokine production induced by polyethylene particles was not substantially affected by the lack of lymphocytes in Pfp/Rag2 double knock out mice. All of these findings are similar to our observations of osteolysis induced by titanium particles. These results provide strong support for both of our hypotheses: that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles and that lymphocytes do not play a major role in particle-induced osteolysis.

The incorporation of different sorts of cancellous bone graft and the reaction of the host bone. A histomorphometric study in sheep.

We performed a morphological and histomorphometric analysis of the use of either autografts, or of frozen or freeze-dried cancellous bone allografts in sheep. A cancellous bone defect was created in the lateral portion of the distal epiphysis of the left femur. Four groups of six animals were monitored for 3 months. In the first group, the cavity was filled with autograft, in the second with frozen allograft and in the third with freeze-dried allograft. In the last group, the cavity was not filled and served as control. A study of the host bone showed that the mean trabecular width of the peripheral osteoid was greatest in the control group, while the number of osteoblasts and osteoclasts was significantly lower in the freeze-dried allograft group. However, the different bone grafts that were used to fill the cavity showed a greater trabecular width and area in the autografts. Among the frozen allografts, these measurements were also greater than in the freeze-dried allograft group. The "erosion surface" of the freeze-dried allograft group was also found to be three times greater, and there were a larger number of osteoclasts and osteoclastic nuclei. We concluded that the "lyophilised" allografts were re-absorbed rapidly and that there were no major morphological differences between the frozen allografts and the autograft groups.

Histomorphologic features of the nasal cavity of pigs exposed to Pasteurella multocida type-D dermonecrotic toxin

Summary Microscopic examination of the nasal mucosa of clinically normal specific-pathogen-free pigs and of toxicogenic type-D Pasteurella multocida toxin challenge-exposed_ specific-pathogen-free pigs indicated that the surface epithelium in pigs of both groups was microscopically normal; erosions or appreciable inflammatory changes were not evident. In pigs of both groups and in all 3 regions of the nasal cavity, the endothelial lining of all blood vessels appeared normal without detectable changes to the walls at postinoculation day 10. Vascular injury in the cartilage or the bone was not discernible in control or challenge-exposed pigs. There were marked differences in the osseous structures of the conchae when the 2 groups were compared. In control pigs, active bone formation and remodeling were observed, and the septal cartilage was normal. In toxin challenge-exposed pigs, there likewise was normal bone formation and remodeling in the vestibular region, and the septal cartilage was normal. In marked contrast, conspicuous changes were observed in the osseous core of the conchae of the respiratory and, sometimes, the olfactory regions. These changes consisted of bone necrosis and resorption by large numbers of osteoclasts with variable replacement by dense mesenchymal stroma, which resulted in conchal atrophy. In the absence of any discernible damage or injury (angiopathy) to the nasal vessels, it appears that the action of the dermonecrotoxin of P multocida serotype D is on the most active osteoblasts and the associated organic matrix of the bone, with subsequent disruption of normal bone formation and remodeling of the nasal conchae.

An immunocytochemical method for studying the kinetics of osteoclast nuclei on intact mouse parietal bone

An immunocytochemical method using an antibody against 5-bromo-2'-deoxyuridine has been applied to the study of the kinetics of osteoclast nuclei on intact mouse parietal bones. Osteoclasts containing tartrate-resistant acid phosphatase show nuclei that are positive for the thymidine analogue within 24 hours of injection into four-day old mice. Labelled osteoclast nuclei decline in number with a half-life of 1.3 days, compatible with a random mechanism of cell death rather than a fixed lifespan. This is shorter than has previously been reported and the possible reasons for this are suggested. The main advantages compared with autoradiography are the shortened processing time and the large number of osteoclasts that can be examined per parietal bone.

Isolation of avian osteoclasts: Improved techniques to preferentially purify viable cells

Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable. The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (greater than 99%) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.

Morphologic features of bone in human osteopetrosis

Trabecular bone biopsies obtained from six patients with malignant osteopetrosis, one patient with benign osteopetrosis, and two controls were examined by light and electron microscopy. Osteopetrotic osteoclasts showed little to no signs of active involvement in bone resorption. Ruffled borders and clear zones were absent in most cells. In all patients there were large numbers of osteoclasts. Numbers of osteoblasts, bone lining cells, and bone marrow stromal cells were extremely low in all patients with malignant osteopetrosis. In six of the patients a mineralized layer of amorphous organic material lacking collagen fibrils was seen covering large areas of the bone or cartilage matrix. We suggest that this layer represents a pathological calcification on which subsequently organic material has accumulated. The abnormalities in osteopetrotic bone are discussed in the light of the pathogenesis of this disease.