Chondrocytes supplemented to bone graft-containing scaffolds expedite cranial defect repair.

Critical maxillofacial bone fractures do not heal spontaneously, thus, often there is a need to facilitate repair via surgical intervention. Gold standard approaches, include the use of autologous bone graft, or devices supplemented with osteogenic growth factors and bone substitutes. This research aimed to employ a critical size calvaria defect model, to determine if the addition of chondrocytes to collagen-containing bone graft substitute, may expedite bone repair. As such, using a critical size rat calvaria defect, we implanted a collagen scaffold containing bone graft substitute (i.e., Bone graft scaffold, BG) or BG supplemented with costal chondrocytes (cBG). The rats were subjected to live CT imaging at 1, 6, 9, and 12 weeks following the surgical procedure and sacrificed for microCT imaging of the defect site. Moreover, serum markers and histological evaluation were assessed to determine osseous tissue regeneration and turnover. Live CT and microCT indicated cBG implants displayed expedited bone repair vs, BG alone, already at 6 weeks post defect induction. cBG also displayed a shorter distance between the defect edges and greater mineral apposition distance compared to BG. Summerizing, the data support the addition of chondrocytes to bone substitute, accelerates the formation of new bone within a critical size defect.

Repairing a critical cranial defect using WISP1-pretreated chondrocyte scaffolds.

In cranial flat bone fractures, spontaneous bone repair will occur only when the fracture ends are in close contact. However, in cases wherein bone discontinuity is extensive, surgical interventions are often required. To this end, autologous bone is harvested and surgically integrated into the site of fracture. Here we propose to use cartilage, as an alternative autologous source, to promote cranial fracture repair. The advantage of this approach is the potential reduction in donor site morbidity, likely due to the avascular and aneural nature of cartilage. As a first step we attempted to induce cartilage mineralization in vitro, using micromass primary chondrocyte cultures, incubated with BMP2 and/or WISP1, which were examined histologically following a 3-week culture period. Next, chondrocyte seeded collagen scaffolds were evaluated in vitro for expression profiles and ALP activity. Finally, chondrocyte-seeded collagen scaffolds were implanted in a Lewis rats 8 mm critical calvaria defect model, which was imaged via live CT for 12 weeks until sacrifice. End points were analyzed for microCT, histology, and serum levels of bone related markers. Micromass cultures exhibited an osseous inducing trend following WISP1 administration, which was maintained in chondrocyte seeded scaffolds. Accordingly, in vivo analysis was carried out to assess the impact of WISP1-pretreated chondrocytes (WCS) versus untreated chondrocytes (UCS) in calvaria defect model and compared to untreated control comprised of a defect-associated blood clot (BC) or empty collagen scaffold (CS) implant. Live CT and microCT exhibited higher mineralization volumes in critical defect implanted with UCS, with some structural improvements in WCS. Histological analysis exhibited higher anabolic bone formation in WCS and trabecular bone was detected in WCS and UCS groups. Chondrocytes implanted into critical cranial defect expedite the formation of native-like osseous tissue, especially after WISP1 priming in culture. Ultimately, these data support the use of autologous chondrocytes to repair critical maxillofacial defects.

The impact of alloplast and allograft on bone homeostasis: Orthodontic tooth movement into regenerated bone.

BACKGROUND: The aim of the study is to examine bone healing following augmentation with allograft or ß-tricalcium phosphate (ß-TCP) and evaluate orthodontic tooth movement (OTM) into the augmented sites. METHODS: The study included two parts. Part I included the alveolar bone regeneration model. Osseous defects were created by extraction of the maxillary first molars in C57BL/6 mice, and the sockets were filled with allograft, ß-TCP, or left unfilled (n = 6/group). Mouse allograft was prepared by a novel method using long bones. Maxillae were collected at 2, 4, and 6 weeks for microcomputed tomography (µCT) and histological analysis. In Part II, OTM was performed after full bone healing, through grafted and unfilled sockets (n = 10/group), and the second molar shift was assessed using µCT. RESULTS: Bone volume and trabeculation were reduced in ß-TCP compared with allograft and non-grafted groups at 2 and 4 weeks post-grafting, but similar at 6 weeks. Graft particles could be detected at 2 weeks post-grafting for ß-TCP, and at 2 and 4 weeks for allograft. Increased osteoclasts' presence was observed in the ß-TCP group at 2 and 4 weeks compared with allograft and control. OTM was similar in the two graft groups, but impaired versus the non-grafted controls. CONCLUSION: ß-TCP and allograft induce full normal healing but alter OTM into the regenerated sites.

Isolation and characterization of mesenchymal stromal progenitors from the temporomandibular joint disc.

Disorders of the temporomandibular joint (TMJ) complex affect 6-12% of the population; the joint's disc is usually involved. Tissue engineering and regenerative medicine may constitute a promising therapeutic approach, with resident stromal progenitor cells a key factor in the process. We hypothesized that the TMJ disc (TMJD) contains multipotent stromal progenitors that may play an important role in regeneration of the disc. TMJD cells were cultured and evaluated for growth kinetics and colony-forming units (CFUs). Single cell-derived clones were isolated and induced to differentiate toward the osteogenic, adipogenic and chondrogenic lineages by culturing in various induction media. Flow cytometry was used to identify multipotent stromal cell surface markers in additional cell samples, and reverse transcription-polymerase chain reaction (RT-PCR) was used to determine gene expression patterns within isolated cells. High numbers of CFUs were observed, indicating cell self-renewal. Biochemical assays showed significantly higher alkaline phosphatase (ALP) activity, lipid droplet concentration and glycosaminoglycan levels in cells cultured in osteogenic, adipogenic and chondrogenic induction medium, respectively. Approximately 1% of the total cell population demonstrated the capability to differentiate into all three mesenchymal lineages. Chondrogenic gene levels within TMJD-derived cells were significantly reduced in passaged culture. Our results support the hypothesis that multipotent stromal progenitor cells populate the TMJD and possess proliferation and differentiation capabilities. These cells may contribute to the regeneration potential of dysfunctional tissue and become the primary component in future attempts at tissue engineering or regeneration of this complex. Copyright © 2015 John Wiley & Sons, Ltd.

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides.

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a "shoulder" sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-µM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as (•)NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.

The effect of a nitroxide antioxidant on ischemia-reperfusion injury in the rat in vivo hind limb model.

Microsurgical procedures such as free tissue transfer or replantations of amputated digits involve an obligatory ischemic period leading to regional tissue oedema, rhabdomyolysis, systemic acidosis, hypercalcemia and multiple organ dysfunction syndrome reflecting ischemia-reperfusion (I/R) injury. Since nitroxide stable radicals act as antioxidants their potential protective effects were tested. Anaesthetized Sabra rats were subjected to regional ischemia of the hind limb for 2 h using a tourniquet. Upon reperfusion rats were injected with 4-OH-2,2,6,6-tetramethylpiperidine-1-oxyl (TPL). Systemic I/R-induced damage was assessed by sampling blood for differential count, lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) serum levels. Regional injury was evaluated by analysing excised muscle samples for oedema (tissue water content) and inflammatory infiltrate (number of cell nuclei in histomorphometric analysis). I/R-induced changes of biomarkers reflecting systemic damage peaked about 8 h following the start of reperfusion and fully disappeared as the biomarkers relaxed to their pre-ischemic values after 24 h. TPL facilitated the recovery of some of these parameters and partially affected release of cellular CPK and LDH. The parameters of I/R-induced regional tissue injury did not demonstrate any recovery and were not inhibited by TPL.

The clinical characteristics of condylar hyperplasia: experience with 61 patients.

PURPOSE: Much reported variation and discord exist regarding mandibular condylar hyperplasia (CH). This study evaluated some of the characteristics of this disorder in a series of 61 patients with active CH. PATIENTS AND METHODS: A total of 61 patients with active temporomandibular CH who had been evaluated in our departments were included. Demographic, clinical, radiologic, and bone scintiscan data were collected and analyzed. Asymmetries were classified as transverse, vertical, or combined. RESULTS: CH was diagnosed during the growth period in 22 patients, and 39 patients were older than 20 years (range, 11 to 80 years). In 66% of the patients, the main complaint was progressive facial asymmetry; and in the remainder, the main complaint was pain, dysfunction, or both. Transverse asymmetry predominated (52%), and vertical or combined asymmetry occurred in 31% and 16% of patients, respectively; asymmetry type was independent of age. The occlusal plane deviated in 48% of the patients. Laterality was significantly gender-biased (females, 72% right; males, 64% left; P = .017). The condylar head shape was normal in 15% of patients, deformed in 27%, and enlarged in 58%; the condylar neck was elongated in 69% and enlarged in 19%. All of these changes were uncorrelated with the type of asymmetry (vertical, transverse, or combined). CONCLUSIONS: CH may occur at any age and is more prevalent in females. Clinicians should be aware that only some patients complain primarily of facial asymmetry, and that symptoms of temporomandibular disease also may be present. Because there is no correlation between the radiologic findings and the clinical evaluation, classification should be simplified and based on clinical manifestation only--in other words, the direction of asymmetry.

Induced osteogenesis by periosteal distraction.

PURPOSE: The purpose of this project was to evaluate a novel technique for inducing osteogenesis through periosteal distraction in a rabbit model. MATERIALS AND METHODS: A periosteal distraction device was rigidly fixed to the lateral surface of the mandible in 10 adult rabbits. Periosteal distraction was started 7 days after placement of the periosteal distraction device. The periosteum was distracted 7 mm over 15 days. The unoperated, contralateral side of the mandible served as the control. The animals were killed at postoperative days 28, 35, 42, and 56. The specimens were then fixed, decalcified, and stained with hematoxylin and eosin. Histologic examination and histomorphometric analysis were performed on all specimens. RESULTS: Nine of 10 periosteal distraction devices remained rigidly fixed to the lateral surface of the mandible. On postoperative day 28, the histologic specimen from the experimental side showed periosteal proliferation and an increase in the number of osteoblasts. On postoperative days 35, 42, and 56, the experimental side showed an increase in the number of osteocytes per unit area, collagen fibers parallel to the vector of distraction, islands of osteoblasts surrounded by newly formed bone, and maturation of bone. An average of 2.86 +/- 0.56 mm of new bone height was formed. CONCLUSION: We report on a novel technique for generating bone by periosteal distraction. Our histologic analysis showed proliferation of the periosteum, an increase in the number of osteoblasts and osteogenesis.

The implications of a broken needle in the pterygomandibular space: clinical guidelines for prevention and retrieval.

The implication of a broken anesthetic injection needle in the posterior part of the oral cavity is described. Needle breakage is preventable if proper preventive measures are used during local anesthesia administration. A broken needle should be removed immediately after a thorough localization and not left in the tissue, as previously believed. Computerized tomography (CT) scan is the proper diagnostic device to locate a broken needle. A careful surgical approach under general anesthesia is recommended to retrieve the needle. Clinical preventive guidelines are described and presented to the pediatric dentist.