Bone Tissue Engineering (BTE) of the Craniofacial Skeleton, Part II: Translational Potential of 3D-Printed Scaffolds for Defect Repair.

Computer-aided design/computer-aided manufacturing and 3-dimensional (3D) printing techniques have revolutionized the approach to bone tissue engineering for the repair of craniomaxillofacial skeletal defects. Ample research has been performed to gain a fundamental understanding of the optimal 3D-printed scaffold design and composition to facilitate appropriate bone formation and healing. Benchtop and preclinical, small animal model testing of 3D-printed bioactive ceramic scaffolds augmented with pharmacological/biological agents have yielded promising results given their potential combined osteogenic and osteoinductive capacity. However, other factors must be evaluated before newly developed constructs may be considered analogous alternatives to the "gold standard" autologous graft for defect repair. More specifically, the 3D-printed bioactive ceramic scaffold's long-term safety profile, biocompatibility, and resorption kinetics must be studied. The ultimate goal is to successfully regenerate bone that is comparable in volume, density, histologic composition, and mechanical strength to that of native bone. In vivo studies of these newly developed bone tissue engineering in translational animal models continue to make strides toward addressing regulatory and clinically relevant topics. These include the use of skeletally immature animal models to address the challenges posed by craniomaxillofacial defect repair in pediatric patients. This manuscript reviews the most recent preclinical animal studies seeking to assess 3D-printed ceramic scaffolds for improved repair of critical-sized craniofacial bony defects.

Mitochondrial Complex I Inhibition in Dopaminergic Neurons Causes Altered Protein Profile and Protein Oxidation: Implications for Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis in a dopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.

Bone Tissue Engineering (BTE) of the Craniofacial Skeleton, Part I: Evolution and Optimization of 3D-Printed Scaffolds for Repair of Defects.

Bone tissue regeneration is a complex process that proceeds along the well-established wound healing pathway of hemostasis, inflammation, proliferation, and remodeling. Recently, tissue engineering efforts have focused on the application of biological and technological principles for the development of soft and hard tissue substitutes. Aim is directed towards boosting pathways of the healing process to restore form and function of tissue deficits. Continued development of synthetic scaffolds, cell therapies, and signaling biomolecules seeks to minimize the need for autografting. Despite being the current gold standard treatment, it is limited by donor sites' size and shape, as well as donor site morbidity. Since the advent of computer-aided design/computer-aided manufacturing (CAD/CAM) and additive manufacturing (AM) techniques (3D printing), bioengineering has expanded markedly while continuing to present innovative approaches to oral and craniofacial skeletal reconstruction. Prime examples include customizable, high-strength, load bearing, bioactive ceramic scaffolds. Porous macro- and micro-architecture along with the surface topography of 3D printed scaffolds favors osteoconduction and vascular in-growth, as well as the incorporation of stem and/or other osteoprogenitor cells and growth factors. This includes platelet concentrates (PCs), bone morphogenetic proteins (BMPs), and some pharmacological agents, such as dipyridamole (DIPY), an adenosine A 2A receptor indirect agonist that enhances osteogenic and osteoinductive capacity, thus improving bone formation. This two-part review commences by presenting current biological and engineering principles of bone regeneration utilized to produce 3D-printed ceramic scaffolds with the goal to create a viable alternative to autografts for craniofacial skeleton reconstruction. Part II comprehensively examines recent preclinical data to elucidate the potential clinical translation of such 3D-printed ceramic scaffolds.

Osteogenic differentiation and reconstruction of mandible defects using a novel resorbable membrane: An in vitro and in vivo experimental study.

To evaluate the cellular response of both an intact fish skin membrane and a porcine-derived collagen membrane and investigate the bone healing response of these membranes using a translational, preclinical, guided-bone regeneration (GBR) canine model. Two different naturally sourced membranes were evaluated in this study: (i) an intact fish skin membrane (Kerecis Oral®, Kerecis) and (ii) a porcine derived collagen (Mucograft®, Geistlich) membrane, positive control. For the in vitro experiments, human osteoprogenitor (hOP) cells were used to assess the cellular viability and proliferation at 24, 48, 72, and 168 h. ALPL, COL1A1, BMP2, and RUNX2 expression levels were analyzed by real-time PCR at 7 and 14 days. The preclinical component was designed to mimic a GBR model in canines (n = 12). The first step was the extraction of premolars (P1-P4) and the 1st molars bilaterally, thereby creating four three-wall box type defects per mandible (two per side). Each defect site was filled with bone grafting material, which was then covered with one of the two membranes (Kerecis Oral® or Mucograft®). The groups were nested within the mandibles of each subject and membranes randomly allocated among the defects to minimize potential site bias. Samples were harvested at 30-, 60-, and 90-days and subjected to computerized microtomography (µCT) for three-dimensional reconstruction to quantify bone formation and graft degradation, in addition to histological processing to qualitatively analyze bone regeneration. Neither the intact fish skin membrane nor porcine-based collagen membrane presented cytotoxic effects. An increase in cell proliferation rate was observed for both membranes, with the Kerecis Oral® outperforming the Mucograft® at the 48- and 168-hour time points. Kerecis Oral® yielded higher ALPL expression relative to Mucograft® at both 7- and 14-day points. Additionally, higher COL1A1 expression was observed for the Kerecis Oral® membrane after 7 days but no differences were detected at 14 days. The membranes yielded similar BMP2 and RUNX2 expression at 7 and 14 days. Volumetric reconstructions and histologic micrographs indicated gradual bone ingrowth along with the presence of particulate bone grafts bridging the defect walls for both Kerecis Oral® and Mucograft® membranes, which allowed for the reestablishment of the mandible shape after 90 days. New bone formation significantly increased from 30 to 60 days, and from 60 to 90 days in vivo, without significant differences between membranes. The amount of bovine grafting material (%) within the defects significantly decreased from 30 to 90 days. Collagen membranes led to an upregulation of cellular proliferation and adhesion along with increased expression of genes associated with bone healing, particularly the intact fish skin membrane. Despite an increase in the bone formation rate in the defect over time, there was no significant difference between the membranes.

Employing Indirect Adenosine 2A Receptors (A2AR) to Enhance Osseointegration of Titanium Devices: A Pre-Clinical Study.

The present study aimed to evaluate the effect of dipyridamole, an indirect adenosine 2A receptors (A2AR), on the osseointegration of titanium implants in a large, translational pre-clinical model. Sixty tapered, acid-etched titanium implants, treated with four different coatings ((i) Type I Bovine Collagen (control), (ii) 10 µM dipyridamole (DIPY), (iii) 100 µM DIPY, and (iv) 1000 µM DIPY), were inserted in the vertebral bodies of 15 female sheep (weight ~65 kg). Qualitative and quantitative analysis were performed after 3, 6, and 12 weeks in vivo to assess histological features, and percentages of bone-to-implant contact (%BIC) and bone area fraction occupancy (%BAFO). Data was analyzed using a general linear mixed model analysis with time in vivo and coating as fixed factors. Histomorphometric analysis after 3 weeks in vivo revealed higher BIC for DIPY coated implant groups (10 µM (30.42% ± 10.62), 100 µM (36.41% ± 10.62), and 1000 µM (32.46% ± 10.62)) in comparison to the control group (17.99% ± 5.82). Further, significantly higher BAFO was observed for implants augmented with 1000 µM of DIPY (43.84% ± 9.97) compared to the control group (31.89% ± 5.46). At 6 and 12 weeks, no significant differences were observed among groups. Histological analysis evidenced similar osseointegration features and an intramembranous-type healing pattern for all groups. Qualitative observation corroborated the increased presence of woven bone formation in intimate contact with the surface of the implant and within the threads at 3 weeks with increased concentrations of DIPY. Coating the implant surface with dipyridamole yielded a favorable effect with regard to BIC and BAFO at 3 weeks in vivo. These findings suggest a positive effect of DIPY on the early stages of osseointegration.

Loss of Notch signaling in skeletal stem cells enhances bone formation with aging.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

Patient-specific 3D printed Poly-ether-ether-ketone (PEEK) dental implant system.

Fused Filament Fabrication (FFF)-based 3D printing is an efficient technique for developing medical implants, but it is not very useful in developing small yet mechanically robust design-specific fixtures such as dental implants (<15 mm). Specifically, it is challenging to 3D print robust Polyetheretherketone (PEEK) small implants due to PEEK's high melting temperature and melt viscosity. However, in this study, we efficiently utilize high-temperature FFF to develop the first-of-its-kind patient-specific robust PEEK dental implants with high print resolution. Specifically, we explore the effects of critical FFF processing conditions on the mechanical properties of the implants and subsequently determine an optimized set of processing conditions that are essential in developing durable dental implant systems. Our results indicate that the 3D printed dental implants exhibit good fatigue properties and suffice the clinical and industrial requirements for dental implants. Furthermore, we prove that the 3D printed implants exhibit adequate mechanical durability even after simulated (accelerated) aging of 30 years.

Radioprotective effect of sesamol on gamma-radiation induced DNA damage, lipid peroxidation and antioxidants levels in cultured human lymphocytes.

Sesamol pretreated (1, 5 and 10 microg/ml) lymphocytes were exposed to different doses of gamma-radiation, i.e., 1, 2 and 4 Gray (Gy) and the cellular changes were estimated by using cytokinesis blocked micronucleus assay (MN), dicentric aberration (DC), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Radiation significantly increased MN, DC frequencies, TBARS levels and decreased GSH and antioxidant enzyme levels in a dose dependent manner. The highest damage to lymphocytes was observed at 4 Gy irradiation. On the other hand, sesamol pretreatment significantly decreased MN, DC frequencies, TBARS levels and increased GSH levels and SOD, CAT and GPx activities in a concentration dependent manner. At 1 Gy irradiation all concentrations of sesamol (1, 5 and 10 microg/ml) significantly protects the lymphocytes from radiation damage. At 2 Gy irradiation 5 and 10 microg/ml of sesamol shows significant radioprotection. Since the highest damage was observed at 4 Gy irradiation both 1 and 5 microg/ml of sesamol pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10 microg/ml of sesamol significantly (p<0.05) protects the lymphocytes from radiation effect. Thus, sesamol pretreatment gives significant protection to cultured human lymphocytes against gamma-radiation induced cellular damage. The possible mechanism involved in the radioprotective influence of sesamol is discussed.

Splenic tumours--autopsy study of ten years.

Human spleen, though being the largest component of reticuloendothelial system, is a very rare site of tumor metastases. Splenic metastases are usually seen as part of multi-organ involvement. Autopsy study conducted over a period of 10 years revealed that the incidence of neoplastic involvement of spleen was 1.45% (70/4812). Primary malignant involvement of spleen was also noted to be a rare entity in present study.

Inducible protective processes in animal systems. III. Adaptive response of meiotic cells of the grasshopper, Poecilocerus pictus, to a low dose of ethyl methanesulfonate.

Meiotic cells of Poecilocerus pictus exposed to a low dose of 0.03 M of ethyl methanesulfonate (EMS) were found to be resistant to the induction of chromosomal anomalies by a subsequent challenge dose (0.12 M) of the same mutagen as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromosomal anomalies in metaphase I and II and anaphase I and II. These results indicate the presence of an inducible chromosomal repair mechanism in meiotic cells of P. pictus. The incidence of chromosome damage was found to be less when the time lag between the conditioning and challenging doses was reduced, suggesting that under the conditions tested, the efficacy of repair enzymes gradually decreases as the time between the two doses increases.

Evidence for a receptor-mediated endocytosis of Alu I in Chinese hamster ovary cells.

Pretreatment of Chinese hamster ovary cells with proteases or with NaN3 leads to less chromosomal aberrations when the cells are posttreated with Alu I compared to the treatment of cells with Alu I alone. The same result is obtained when the cells are treated with Alu I at 0 degree C instead of 37 degrees C. The cells recover from the protease treatment when they are kept in medium before treatment with Alu I. These results are interpreted to mean that Alu I is bound by surface receptors and that the Alu I-receptor complexes are internalized by an energy-dependent endocytotic process.

Effect of heat treatment on chromosomal aberrations induced by the alkylating agent trenimon or the restriction endonuclease Alu I in Chinese hamster ovary (CHO) cells.

Heat treatment of CHO cells in the G1-phase of the cell cycle leads to chromatid-type aberrations in first posttreatment metaphases. Posttreatment of heat-treated cells with the alkylating agent trenimon leads to a synergistic effect on the production of chromatid-type exchanges. These results indicate that heat induces lesions which like the lesions produced by trenimon give rise to chromatid-type aberrations during the first posttreatment S-phase, and that these lesions can interact with each other to produce chromatid-type exchanges. Treatment of CHO cells in the G1-phase of the cell cycle with the restriction endonuclease Alu I induces chromosomal aberrations. Pretreatment of cells with heat leads to a reduction of Alu I induced chromosome-type aberrations. When cells are allowed to recover after heat treatment for 22 h, the aberration frequencies produced by Alu I are the same as in cells not treated with heat. These findings can be explained by assuming that heat-induced accumulation of accessory proteins in the chromatin protects the DNA from being cut by Alu I, and that the cells recovered from the heat-induced protein accumulation after 22 h.

Liquid-holding experiments with human lymphocytes. III. Experiments with G0 and G1 cells.

Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the G0 (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sister-chromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in G0 cells and that G1 cells can repair both DEB and MNU induced lesions.

In vivo cytogenetic analyses of the carbamate pesticides Dithane M-45 and Baygon in mice.

Two carbamate pesticides, Dithane M-45 and Baygon, were analysed for their cytogenetic effects using meiotic chromosome analysis and the micronucleus test in Swiss albino male mice. The three sub-lethal doses of 1687.5, 3375 and 5962.5 mg/kg bw of Dithane M-45 and 1250, 2500 and 3750 mg/kg bw of Baygon were employed in all the experiments. The results demonstrate that neither Dithane M-45 nor Baygon could induce a significant (P > 0.05) increase in the number of chromosomal aberrations in the germ cells or in the percentage of micronuclei in erythrocytes.

Induction of chromosomal aberrations in Chinese hamster ovary cells by the restriction endonuclease Dra I: dose-effect relationships and effect of substitution of chromosomal DNA with bromodeoxyuridine.

Treatment of G1-phase Chinese hamster ovary (CHO) cells with the restriction endonuclease Dra I (recognition site TTT/AAA) leads to the induction of chromosome-type aberrations. The dose-effect relationships or the frequencies of polycentric chromosomes have a strong linear component. Prelabelling of the cells with bromodeoxyuridine (B) leads to a strong suppression by the chromosome breaking activity of Dra I. This may be explained by assuming that substitution of T by B renders the recognition site of Dra I resistant to being cut by the enzyme.

Fine-needle aspiration cytology in lymphadenopathy of HIV-positive patients.

Fine-needle aspiration cytology (FNAC) of 32 HIV-positive cases presenting with lymphadenopathy was performed to evaluate its role in this group of patients. For each case air-dried smears were stained with Leishman, hematoxylin and eosin, and Zeihl-Neelsen stains for acid fast bacilli (AFB). The results were tuberculous (TB) lymphadenopathy (15), reactive lymphadenopathy (10), acute lymphadenitis/abscess (5), and suspected malignancy (2). In seven cases of TB lymphadenitis findings were suggestive of TB since no AFB was demonstrable on the cytology smears. In TB lymphadenitis, two additional patterns besides necrotising granulomatous (4) and granulomatous (2) were observed. These were necrotising (6) and necrotising suppurative (3) patterns. FNAC is a simple, inexpensive, rapid investigative procedure which can reduce surgical excisions and provide definite guidelines about further management.

Inducible protective processes in animal systems: adaptive response to a low dose of methyl methanesulfonate in mouse bone marrow cells.

To investigate the induction of adaptive response (inducible protective processes) in mitotic cells of Swiss albino mouse, a monofunctional alkylating agent methyl methanesulfonate (MMS) was employed. When the animals treated with a low dose of 50 mg/kg body weight were challenged with a subsequent high (challenging) dose of 150 mg/kg body weight, after different time lags (2,5,8 or 10 hr), the yield of chromosomal aberrations in bone marrow cells was found to be significantly reduced compared to the additive effects of both conditioning and challenging doses. It seems, therefore, that the low dose of MMS employed has made the cells less sensitive against further clastogenic effect of challenge dose of MMS. The data clearly suggest that the phenomenon of adaptive response to methylating agents can be encountered in in vivo mammalian cells. Furthermore, it is also observed that ethylating agent EMS is a poor inducer of adaptive response than its corresponding methylating agent MMS in the bone marrow cells of mouse.

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes.

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.