Number Of Empty Lacunae Research Articles

Evaluation of Methenolone Enanthate Efficacy in Preventing MRONJ: A Randomized Trial on Rats.

It is well-known that oral surgical procedures pose a high risk for medication-related osteonecrosis of the jaw in patients taking bisphosphonates. Although some position papers and guidelines have been published with regard to its treatment, few studies have investigated prevention methods. This study investigates the effectiveness of methenolone enanthate, an anabolic steroid, for the prevention of medication-related osteonecrosis of the jaw. Thirty-six Wistar rats were divided into three groups. Two experimental groups, Z and ZM, took zoledronic acid for 6 weeks prior to extraction of the left maxillary first molar. The Group ZM also was given methenolone enanthate continuously for 1 week before and 4 weeks after the extraction. The control group was not given any medication. The rats were euthanized 5 weeks after extraction. The extraction socket was evaluated clinically for bone exposure and histologically for inflammation, hyperemia, collagen fibers, epithelialization, number of osteoclasts, and empty lacunae. Six rats died during the experimental research. The bone exposure rate, mean numbers of attached osteoclasts (in 40× magnification), and empty lacunae (in 100× magnification) were 0%, 4%, and 0.8% in Group C; 75%, 1%, and 8% in Group Z; and 10%, 2.1%, and 3% in Group ZM, respectively. Significant differences exist between all groups regarding the number of empty lacunae. There were significant differences between Group C/ZM and Group Z in terms of bone exposure rate, inflammation, hyperemia, collagen fiber organization, and epithelialization. In our tested preclinical model, methenolone enanthate has shown potential for preventing medication-related osteonecrosis of the jaw.

Histological features of knee osteoarthritis treated with triamcinolone acetonide and hyaluronic acid.

Osteoarthritis (OA) is one of the most common degenerative joint diseases leading to disability in the end stage. Although intra-articular triamcinolone acetonide (TA) is one of the OA treatments that have been widely used, the side effects of such corticosteroids are still controversial. Intra-articular hyaluronic acid (HA) injection is another therapeutic option for patients with OA who do not want to use corticosteroids because of their side effects. However, the difference between the histological features associated with TA and HA in the treatment of OA remains unclear. Thus, the present study aimed to compare the histological effects of TA and HA on the cartilage of patients with knee OA. In the current study, 31 patients diagnosed with grade 3-4 knee OA on the Kellgren-Lawrence radiographic grading scale were separated into three groups: TA (n=12); HA (n=7) and untreated group (n=12). Histological examination of the whole articular cartilages of the patients was performed with hematoxylin and eosin and Alcian staining, as well as using a TUNEL assay. Clinical data such as cartilage thickness, structural and component deterioration, proteoglycan levels, apoptosis and empty lacunae were compared between the three groups. The results showed a high level of deterioration in both TA and HA groups but not in the untreated group, although the thickness of cartilage in the HA group was lower compared with that in the TA and untreated groups. The proteoglycan levels in the TA group were lower compared with those in the HA group. Moreover, the number of empty lacunae in the HA group was higher compared with that in the TA group, while no difference in apoptosis was found between TA and HA groups. A significant difference was not found in the histological staining between TA and HA groups. On the other hand, a significant difference was found in cartilage deterioration between the medial and lateral sides in these groups. TA and HA groups showed comparable histological results. TA injection is cheaper and easier but has more adverse effects for patients with knee OA than HA injection. Therefore, orthopaedists should select TA or HA based on the economic and specific needs of patients.

Complementarity of surgical therapy, photobiomodulation, A-PRF and L-PRF for management of medication-related osteonecrosis of the jaw (MRONJ): an animal study

BackgroundThis study aimed to evaluate the complementarity of surgical therapy, photobiomodulation (PBM), advanced platelet-rich fibrin (A-PRF), and Leukocyte and platelet-rich fibrin (L-PRF) for the management of medication-related osteonecrosis of the jaw (MRONJ).MethodsSixty rats underwent injection of zoledronate followed by left mandibular first and second molar extractions to induce MRONJ lesions. All rats were examined for the signs of MRONJ 8 weeks post-dental extraction. Forty-nine rats with positive signs of MRONJ were appointed to seven different groups as follows: control (Ctrl); surgery alone (Surg); surgery and PBM (Surg + PBM); surgery and A-PRF insertion (Surg + APRF); surgery and L-PRF insertion (Surg + LPRF); surgery, A-PRF insertion, and PBM (Surg + APRF + PBM); surgery, L-PRF insertion, and PBM (Surg + LPRF + PBM). Euthanasia was carried out 30 days after the last treatment session. The lesions' healing was evaluated clinically, histologically, and radiographically. Data were analyzed using STATA software version 14, and the statistical significance level was set at 5% for all cases.ResultsAccording to the present study, A-PRF and L-PRF treatment resulted in significant improvements in clinical, histological, and radiographical parameters compared to the Ctrl group (P < 0.05). The PBM also decreased wound dimensions and the number of empty lacunae compared to the Ctrl group (P < 0.05). Surg + APRF + PBM and Surg + LPRF + PBM were the only groups that presented a significantly higher mean number of osteocytes (P < 0.05). No significant differences were observed between A-PRF and L-PRF treatment groups (P > 0.05).ConclusionsSurgical resection followed by applying A-PRF or L-PRF reinforced by PBM showed optimal wound healing and bone regeneration in MRONJ lesions.

Osteocyte Apoptosis, Bone Marrow Adiposity, and Fibrosis in the Irradiated Human Mandible

PurposeTo assess the effect of radiation therapy on osteocyte apoptosis, osteocyte death, and bone marrow adipocytes in the human mandible and its contribution to the pathophysiology of radiation damage to the mandibular bone. Methods and MaterialsMandibular cancellous bone biopsies were taken from irradiated patients and nonirradiated controls. Immunohistochemical detection of cleaved caspase-3 was performed to visualize apoptotic osteocytes. The number of apoptotic osteocytes per bone area and per total amount of osteocytes, osteocytes per bone area, and empty lacunae per bone area were counted manually. The percentage fibrotic tissue and adipose tissue per bone marrow area, the percentage bone marrow of total area, and the mean adipocyte diameter (μm) was determined digitally from adjacent Goldner stained sections. ResultsBiopsies of 15 irradiated patients (12 men and 3 women) and 7 nonirradiated controls (5 men and 2 women) were assessed. In the study group a significant increase was seen in the number of empty lacunae, the percentage of adipose tissue of bone marrow area, and the adipocyte diameter. There was no significant difference in bone marrow fibrosis nor apoptotic osteocytes between the irradiated group and the controls. ConclusionsIrradiation alone does not seem to induce excessive bone marrow fibrosis. The damage to bone mesenchymal stem cells leads to increased marrow adipogenesis and decreased osteoblastogenic potential. Early osteocyte death resulting in avital persisting bone matrix with severely impaired regenerative potential may contribute to the vulnerability of irradiated bone to infection and necrosis.

Suppression of Bone Necrosis around Tooth Extraction Socket in a MRONJ-like Mouse Model by E-rhBMP-2 Containing Artificial Bone Graft Administration.

Medication-related osteonecrosis of the jaw (MRONJ) is related to impaired bone healing conditions in the maxillomandibular bone region as a complication of bisphosphonate intake. Although there are several hypotheses for the onset of MRONJ symptoms, one of the possible causes is the inhibition of bone turnover and blood supply leading to bone necrosis. The optimal treatment strategy for MRONJ has not been established either. BMP-2, a member of the TGF-β superfamily, is well known for regulating bone remodeling and homeostasis prenatally and postnatally. Therefore, the objectives of this study were to evaluate whether cyclophosphamide/zoledronate (CY/ZA) induces necrosis of the bone surrounding the tooth extraction socket, and to examine the therapeutic potential of BMP-2 in combination with the hard osteoinductive biomaterial, β-tricalcium phosphate (β-TCP), in the prevention and treatment of alveolar bone loss around the tooth extraction socket in MRONJ-like mice models. First, CY/ZA was intraperitoneally administered for three weeks, and alveolar bone necrosis was evaluated before and after tooth extraction. Next, the effect of BMP-2/β-TCP was investigated in both MRONJ-like prevention and treatment models. In the prevention model, CY/ZA was continuously administered for four weeks after BMP-2/β-TCP transplantation. In the treatment model, CY/ZA administration was suspended after transplantation of BMP-2/β-TCP. The results showed that CY/ZA induced a significant decrease in the number of empty lacunae, a sign of bone necrosis, in the alveolar bone around the tooth extraction socket after tooth extraction. Histological analysis showed a significant decrease in the necrotic alveolar bone around tooth extraction sockets in the BMP-2/β-TCP transplantation group compared to the non-transplanted control group in both MRONJ-like prevention and treatment models. However, bone mineral density, determined by micro-CT analysis, was significantly higher in the BMP-2/β-TCP transplanted group than in the control group in the prevention model only. These results clarified that alveolar bone necrosis around tooth extraction sockets can be induced after surgical intervention under CY/ZA administration. In addition, transplantation of BMP-2/β-TCP reduced the necrotic alveolar bone around the tooth extraction socket. Therefore, a combination of BMP-2/β-TCP could be an alternative approach for both prevention and treatment of MRONJ-like symptoms.

Zoledronic Acid Deteriorates Soft and Hard Tissue Healing of Murine Tooth Extraction Sockets in a Dose-Dependent Manner.

The pathophysiology, histopathology, and immunopathology of bisphosphonate-related osteonecrosis of the jaw (BRONJ) Stage 0 remain unclear. The aim of this study was to investigate the effects of high-dose bisphosphonates on tooth extraction socket healing by creating a murine model of BRONJ Stage 0-like lesions using 8-week-old female C57BL/6J mice. Zoledronic acid (Zol) was administered subcutaneously twice a week for 7weeks at doses of 0.1mg/kg/week (moderate dose; Zol-M), 0.5mg/kg/week (high dose; Zol-H1), and 1.0mg/kg/week (higher dose; Zol-H2). Saline was used as a control (VC). Both maxillary first molars were extracted 3weeks after drug treatment. Maxillae, long bones, and sera were collected 4weeks post-extraction (n = 7 mice/group). Microcomputed tomography, histological, immunohistochemical, and ELISA analyses were performed. A ceiling effect for Zol was noted at the Zol-H1 dose. Osseous healing of extraction sites was significantly impaired with increased necrotic bone and the number of empty lacunae in a Zol dose-dependent manner. Zol significantly decreased epithelial thickness, due to a decrease in thickness of the stratum spinosum, in both Zol-H1 and Zol-H2. Both Zol-H1 and Zol-H2 significantly suppressed the distribution of F4/80+ macrophages in the connective tissue of tooth extraction sockets, although gross healing appeared to be normal. Intriguingly, both Zol-H1 and Zol-H2 significantly increased the numbers of TRAP+ mononuclear cells and detached osteoclasts in the connective tissue and bone marrow of extraction sites compared to VC and Zol-M, correlated with serum TRAcP5b levels. The created murine model of BRONJ Stage 0-like lesions becoming more severe in a dose-dependent manner may help to understand the pathophysiology and histopathology of BRONJ Stage 0 in humans.

Establishing rabbit model of osteonecrosis of the femoral head by intraosseous liquid nitrogen freezing method

Objective To establish the rabbit model of osteonecrosis of the femoral head (ONFH) by intraosseous liquid nitrogen freezing method. Methods One side femoral head of eighteen New Zealand rabbits was randomly selected for establishing the rabbit models of ONFH by intraosseous liquid nitrogen freezing method, and the another side as normal controls. After 2, 4, and 8 weeks after operation, gross, radiological and histopathological observations were observed. Results The normal side of the femoral head had no abnormal changes, and the number of empty bone lacunae accounted for (11.02±1.56)%. On the experimental side, X-ray showed that the femoral head density increased at the 2nd week. At 4th week, the cystic changes were seen and bone density increased. At 8th week, the bone density of the femoral head was uneven, and mild collapse of weight bearing area was found in 2 femoral heads. Histopathologically, at 2nd week, hematopoietic tissue decreased, bone trabecular necrosis and bone marrow focal necrosis, and empty bone lacunae increased significantly [(73.33±7.21)%] in the femoral heads. At 4th week, trabecular bone thinning and derangement were found, empty bone lacunae increased significantly [(81.52±7.35)%], and fibrous tissues were seen in the femoral heads. At 8th week, the necrotic area in the femoral head was significantly increased, trabecular bone became thinner, sparse and broken, and a large number of empty lacunae [(88.13±8.12)%] were seen. There were some hyperplastic connective tissue in the necrotic area, and repaired reaction in the peripheral of the necrotic area. There was no damage and stripping of the cartilage surface of the femoral head. On the experimental side, osteonecrosis appeared in the femoral heads. At 2nd, 4th, and 8th week, the lacunae count was significantly greater on the experimental side than that of the normal side (t2nd week=12.168, P2nd week=0.007; t4th week=13.048, P4th week=0.006; t8th week=13.048, P8th week=0.006). Conclusion Establishing the rabbit models of ONFH with intraosseous liquid nitrogen freezing method is simple and easy, which can simulate clinical early stage of ONFH and suitable for the standardizational treatment study. Key words: Liquid nitrogen freezing; Osteonecrosis of the femoral head; Models, animal; Rabbit

Morphological study of cartilage cell death in patients affected by osteoarthritis and chondrocalcinosis

The role of chondrocyte death in the pathogenesis of osteoarthritis (OA) has been largely discussed in literature, but its relative contribution is difficult to assess (1). Chondrocyte death, be it apoptotic, necrotic or chondroptotic, has been clearly documented in OA and a certain correlation between the degree of cartilage damage and chondrocyte apoptosis has been demonstrated (2;3). Conversely, the relationship between the different types of cell death and chondrocalcinosis (CC) is still little known, as well as the presence and role of chondroptotic cells. The aim of this research was to compare chondrocyte behavior in the cartilage of osteoarthritic and chondrocalcinotic knees, evaluating the different types of cell death by means of optical and electron microscopy. During total knee replacement surgeries, cartilage specimens of femoral condyle have been withdrawn and their transversal semithin sections, stained with toluidine blue and alizarin solutions, have been investigated by optical microscopy. From the same samples, thin sections were obtained for transmission electron microscopy to evaluate, at high magnification, the specific ultrastructural features of different types of cell death. Cartilage specimens from both conditions revealed a thickness reduction of superficial layer and a high number of empty lacunae in the middle layer. Calcium pyrophosphate crystals appeared in the samples of patients affected by CC. In osteoarthritic cartilage, numerous chondrocytes revealed necrotic features, whereas, in chondrocalcinotic tissue, the middle zone was characterized by morphological patterns suggestive of chondroptosis, such as chromatin condensation mostly localized at the nuclear periphery, mitochondria alterations, a marked increase in endoplasmic reticulum, the presence of a diffuse autophagic component and the extrusion of cellular material into the lacunae. In conclusion, a different distribution of cell death types seems to characterize the intermediate layers of cartilage specimens from patients affected by CC compared to OA.

Effect of Different Storage Media on the Regenerative Potential of Autogenous Bone Grafts: A Histomorphometrical Analysis in Rabbits

The success of autogenous bone graft is related to the graft cell viability. In bone-grafting procedures, harvested grafts are often maintained in extraoral media while the recipient site is prepared. The aim of this study was to evaluate in vivo the effect of storage media over autogenous bone grafts during the transsurgical time. Two grafts were removed bilaterally from the calvaria of 18 rabbits. One graft was immediately fixed in the mandibular angle (control group), and the other was maintained in air exposure (dry group), 0.9% NaCl solution (saline group), or platelet-poor plasma (PPP group) during 30 minutes and stabilized in the symmetrical location of control grafts. After 28 days, the animals were euthanized and the bone fragments were removed, demineralized, and embedded in paraffin. Histological evaluation was performed under light microscope. Empty lacunae and bone graft area quantification were carried out for the sections. The histomorphometrical analysis revealed reduction of the graft area and increase of empty lacunae in the dry group when compared with control. No significant differences were found in the number of empty lacunae or bone graft area between the saline group and its control and also between the PPP group and its control. The dry group showed more empty lacunae and less graft area than the saline and PPP groups. In accordance with the results, PPP and physiologic solution demonstrated osteocyte preservation and bone graft area maintenance, being satisfactory storage media for autogenous bone grafts during the transsurgical period.

Increased osteocyte death and mineralization inside bone after parathyroidectomy in patients with secondary hyperparathyroidism

In order to gain insight into the mechanisms underlying the dynamic changes in bone metabolism and bone quality after parathyroidectomy (PTX) in secondary hyperparathyroid patients with high levels of parathyroid hormone (PTH), we performed bone histomorphometric analysis with tetracycline labeling in iliac bone biopsy specimens taken before and after PTX, with special attention paid to osteocytes. At 2 to 4 weeks after PTX, PTH concentrations decreased markedly with evident reductions in bone turnover markers. Histomorphometry revealed that at 2 to 4 weeks following PTX, the osteoclast surface decreased to nearly 0%, with a substantial increase in osteoid volume and a reduction in fibrosis volume. Labeling with tetracycline was observed not only at the mineralization front on the bone surface but also around the osteocyte lacunar walls and canaliculi within both the basic multicellular units (BMUs) and bone structural units (BSUs), suggesting that mineralization was taking place along the lacunocanalicular system after PTX. The tetracycline-labeled area was much greater in the BSUs than in the BMUs and at the mineralization front, and the tetracycline labeling in the BSUs was markedly increased after PTX compared with that in the low- and high-PTH control groups without PTX. The osteocyte number was decreased significantly after PTX, concomitant with an increase in the number of empty lacunae and a reduction of lacunar volume. Thus the increased osteocyte death and mineralization around the lacunocanalicular system in association with a rapid decline in PTH may underlie the changes in bone metabolism and quality that occur following PTX.

Increased Osteocyte Apoptosis in Multiple Myeloma Patients: A Potential Role in Bone Remodeling Alterations Related to Osteolytic Bone Lesions.

Abstract 830Osteocytes are non-proliferative differentiated cells of osteogenic lineage located in the bony lacuna/canalicular system of bone and that have been recently hypothesized to regulate local bone remodeling in part through the cell death and apoptosis. A reduction of osteocyte viability has been demonstrated in osteoporotic bone related to estrogen deficiency and glucorticoid administration. As known, multiple myeloma (MM)–induced osteolysis and/or osteoporosis are characterized by severely imbalanced and uncoupled bone remodeling due to increased osteoclast recruitment and suppressed osteoblast differentiation that occur in close contact with MM cells infiltration. Actually, the potential involvement of osteocytes in bone remodeling alterations occurring in MM patients is not known and it has been investigated in the present study.Firstly we performed histological analysis on bone biopsies obtained from iliac crest in a cohort of 34 patients with MM at the diagnosis (ISS I-III, mean age±SD: 72±10), 52% of them with osteolytic bone lesions at the skeletal survey and 10 patients with monoclonal gammopathy of uncertain significance (MGUS) (mean age± SD: 71±14). Sex-aged matched subjects without hematological malignancies or osteoporosis or metabolic bone disease (n°=10) were also analyzed. Histological analyses were performed on Toluidine blu and Gomori's three-chromic stained sections observed under a Light Microscope (LM). On a total of 500 osteocyte lacunae X section we evaluated both the number of viable osteocytes and the number of dead/degenerated/apoptotic osteocytes together with the number of empty lacunae. We found a significant reduction in the percentage of viable osteocytes in MM patients as compared to healthy controls (% of viable osteocytes: mean ± SD: 25±10% vs. 36±9%; p= 0.003) whereas the difference between MM and MGUS did not reach a statistical significance (25±10% vs. 30.5±16%; p= 0.2). Consistently the number of death osteocytes and empty lacunae was significantly increased in MM vs. healthy subjects (mean ± SD/500 lacunae: 371.3± 52 vs. 315±46; p=0.006) but not as compared to MGUS (371.3±52 vs 337±81; p=0.2). As regard the skeletal involvement in MM patients we found that the percent of viable osteocytes was significantly lower in osteolytic vs. non-osteolytic patients (19±9% vs. 30±7%; p= 0.01) as well as the number of death osteocytes and empty lacunae was higher in osteolytic vs. non-osteolytic patients (mean ± SD/500 lacunae: 394±41 vs. 315±55; p=0.04). Following, in order to verify the occurrence of apoptosis both LM and Transmission Electron Microscopy (TEM) observations were also performed. Under LM, TUNEL analysis showed higher presence of apoptotic cells in those specimens obtained from MM patients in comparison with those obtained from healthy controls. Moreover an increase in the number of apoptotic osteocytes was observed in MM with bone lesions as compared to those without osteolysis. These observations were further confirmed in vitro by ultrastructural analysis on mono-layers of human preosteocyte cells (HOB-01-C1) incubated with or without conditioned media (CM) taken from human myeloma cell lines (HMCLs) KMS12 and JJN3 or co-cultured with them. TEM observations, showed cells in apoptosis with apoptotic bodies and degenerated non-apoptotic cells in preosteocytes treated with HMCL-CM or co-cultured with HMCLs as compared to non-treated cells. Interestingly, we also found that CM of preosteocytes co-cultured with HMCLs but not those of non co-cultured cells significantly increased CD14+ -derived osteoclastogenesis evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit-forming assay.In conclusion our data demonstrate that MM bone is characterized by a reduction of viable osteocytes and an increase of osteocyte apoptosis in relation to the presence of bone lesions that may represent a triggering event to the increase of osteoclast recruitment in MM patients. Disclosures:No relevant conflicts of interest to declare.

Morphometric investigation on osteocytes in human auditory ossicles

An osteocyte lacunae differential count (1-lacunae with live osteocytes, 2-lacunae with degenerating osteocytes, 3-empty lacunae) was carried out on ear ossicles and clavicles from cadavers as well as on stapes removed by stapedotomy. The distance of the three types of lacunae from the vascular source was also determined by a computer-assisted light microscope. Results showed that the delayed fixation of bone from cadavers does not significantly interfere with osteocyte preservation, at least with the scope of this investigation. The results of osteocyte differential count show that the number of empty lacunae and lacunae with degenerating osteocytes: (a) is significantly higher in ear ossicles than in clavicles, (b) increases with age, (c) is higher in stapes than in incuses and mallei, (d) increases with the distance from the vascular sources in both ear ossicles and clavicles. Additionally it appeared that the process of osteocyte degeneration in ear ossicles is very rapid and widespread, over 40% of the cells being dead within the 2nd year of age. In the light of the recent literature and personal findings, which ascribe to osteocytes the function of mechanical detectors, and considering that bone remodeling occasionally occurs in ear ossicles, it is postulated that osteocyte death in these bones could be a programmed phenomenon (apoptosis?), due to which they lose the ability to react to strains and stresses and achieve the structural stability they need to perform their peculiar stereotyped function.

An electron microscopic study of the changes observed in osteocytes under ischemic conditions.

The purpose of this study was to observe the process of ischemia in osteocytes using light and electron microscopy and to compare the changes in these ischemic osteocytes with those in other types of osteocytes (i.e., degenerative osteocytes in physiological states, steroid-induced lipid-accumulating osteocytes) that have been previously reported. Five female Japanese white rabbits were used in this study. Osteochondral chips were taken from one side of the femoral condyle, covered with Millipore filters, and then inserted into the other side of the knee joint. These tissues were examined after 12 h and after 2, 5, 8, and 14 days of ischemia under both light and electron microscopy. Under light microscopy, osteocytes and lacunae were classified into four types: normal osteocyte, pyknotic osteocyte, pale osteocyte, and empty osteocyte lacuna. The number of each type of osteocyte (or lacuna) in a settled area was counted. The ratio of normal osteocytes decreased significantly (p less than 0.001) after the second day of ischemia. Pyknotic osteocytes increased at 12 h (p less than 0.01) and 2 days (p less than 0.001) of ischemia. On the fifth day of ischemia, the percentage of pale osteocytes reached a peak. This was followed by a gradual increase in the number of empty lacunae. On the fourteenth day of ischemia, empty lacunae constituted greater than 40% of the cell types. When viewed by electron microscopy, these necrotic osteocytes were similar to the degenerative osteocytes that have been observed in physiological states and apparently different from lipid-accumulating osteocytes. The results suggested that there could be at least two types of necrotic processes in osteocytes that eventually lead to cell death.