Impaired motor unit recovery and maintenance in a knock-in mouse model of ALS-associated Kif5a variant.

Kinesin family member 5A (KIF5A) is an essential, neuron-specific microtubule-associated motor protein responsible for the anterograde axonal transport of various cellular cargos. Loss of function variants in the N-terminal, microtubule-binding domain are associated with hereditary spastic paraplegia and hereditary motor neuropathy. These variants result in a loss of the ability of the mutant protein to process along microtubules. Contrastingly, gain of function splice-site variants in the C-terminal, cargo-binding domain of KIF5A are associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease involving death of upper and lower motor neurons, ultimately leading to degradation of the motor unit (MU; an alpha motor neuron and all the myofibers it innervates) and death. These ALS-associated variants result in loss of autoinhibition, increased procession of the mutant protein along microtubules, and altered cargo binding. To study the molecular and cellular consequences of ALS-associated variants in vivo, we introduced the murine homolog of an ALS-associated KIF5A variant into C57BL/6 mice using CRISPR-Cas9 gene editing which produced mutant Kif5a mRNA and protein in neuronal tissues of heterozygous (Kif5a+/c.3005+1G>A; HET) and homozygous (Kif5ac.3005+1G>A/c.3005+1G>A; HOM) mice. HET and HOM mice appeared normal in behavioral and electrophysiological (compound muscle action potential [CMAP] and MU number estimation [MUNE]) outcome measures at one year of age. When subjected to sciatic nerve injury, HET and HOM mice have delayed and incomplete recovery of the MUNE compared to wildtype (WT) mice suggesting an impairment in MU repair. Moreover, aged mutant Kif5a mice (aged two years) had reduced MUNE independent of injury, and exacerbation of the delayed and incomplete recovery after injury compared to aged WT mice. These data suggest that ALS-associated variants may result in an impairment of the MU to respond to biological challenges such as injury and aging, leading to a failure of MU repair and maintenance. In this report, we present the behavioral, electrophysiological and pathological characterization of mice harboring an ALS-associated Kif5a variant to understand the functional consequences of KIF5A C-terminal variants in vivo.

AAV1.NT-3 gene therapy increases muscle fiber diameter through activation of mTOR pathway and metabolic remodeling in a CMT mouse model.

Neurotrophin 3 (NT-3) has well-recognized effects on peripheral nerve and Schwann cells, promoting axonal regeneration and associated myelination. In this study, we assessed the effects of AAV.NT-3 gene therapy on the oxidative state of the neurogenic muscle from the TremblerJ (Tr J ) mice at 16 weeks post-gene injection and found that the muscle fiber size increase was associated with a change in the oxidative state of muscle fibers towards normalization of the fiber type ratio seen in the wild type. NT-3-induced fiber size increase was most prominent for the fast twitch glycolytic fiber population. These changes in the Tr J muscle were accompanied by increased phosphorylation levels of 4E-BP1 and S6 proteins as evidence of mTORC1 activation. In parallel, the expression levels of the mitochondrial biogenesis regulator PGC1α, and the markers of glycolysis (HK1 and PK1) increased in the TrJ muscle. In vitro studies showed that recombinant NT-3 can directly induce Akt/mTOR pathway activation in the TrkC expressing myotubes but not in myoblasts. In addition, myogenin expression levels were increased in myotubes while p75 NTR expression was downregulated compared to myoblasts, indicating that NT-3 induced myoblast differentiation is associated with mTORC1 activation. These studies for the first time have shown that NT-3 increases muscle fiber diameter in the neurogenic muscle through direct activation of mTOR pathway and that the fiber size increase is more prominent for fast twitch glycolytic fibers.

Follistatin Gene Therapy for Sporadic Inclusion Body Myositis Improves Functional Outcomes.

Sporadic inclusion body myositis, a variant of inflammatory myopathy, has features distinct from polymyositis/dermatomyositis. The disease affects men more than women, most commonly after age 50. Clinical features include weakness of the quadriceps, finger flexors, ankle dorsiflexors, and dysphagia. The distribution of weakness is similar to Becker muscular dystrophy, where we previously reported improvement following intramuscular injection of an isoform of follistatin (FS344) by AAV1. For this clinical trial, rAAV1.CMV.huFS344, 6 × 1011 vg/kg, was delivered to the quadriceps muscles of both legs of six sporadic inclusion body myositis subjects. The primary outcome for this trial was distance traveled for the 6-min walk test. The protocol included an exercise regimen for each participant. Performance, annualized to a median 1-year change, improved +56.0 m/year for treated subjects compared to a decline of -25.8 m/year (p = 0.01) in untreated subjects (n = 8), matched for age, gender, and baseline measures. Four of the six treated subjects showed increases ranging from 58-153 m, whereas two were minimally improved (5-23 m). Treatment effects included decreased fibrosis and improved regeneration. These findings show promise for follistatin gene therapy for mild to moderately affected, ambulatory sporadic inclusion body myositis patients. More advanced disease with discernible muscle loss poses challenges.

VIP-expressing dendritic cells protect against spontaneous autoimmune peripheral polyneuropathy.

The spontaneous autoimmune peripheral polyneuropathy (SAPP) model in B7-2 knockout nonobese diabetic mice mimics a progressive and unremitting course of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, bone marrow-derived dendritic cells (DCs) were transduced to express vasoactive intestinal polypeptide (VIP) using a lentiviral vector (LV-VIP). These transduced DCs (LV-VIP-DCs) were then injected intravenously (i.v.) into 16-week-old (before disease onset) and 21-week-old (after disease onset) SAPP mice in order to prevent or attenuate the disease. Outcome measures included behavioral tests, clinical and histological scoring, electrophysiology, real-time PCR, flow cytometry analyses, and enzyme-linked immunosorbent assay. LV-VIP-DCs were recruited to the inflamed sciatic nerve and reduced the expression of inflammatory cytokines. A single injection of LV-VIP-DC delayed the onset of disease, stabilized, and attenuated clinical signs correlating with ameliorated behavioral functions, reduced nerve demyelination, and improved nerve conduction. This proof-of-principle study is an important step potentially leading to a clinical translational study using DCs expressing VIP in cases of CIDP refractory to standard immunosuppressive therapy.

Comparison of the popliteal artery and the capsule of the posterior knee (IPACK) block and the genicular nerve block in primary total knee arthroplasty: A prospective randomized trial.

OBJECTIVES: To compare the efficacy of genicular block and interspace between the popliteal artery and the posterior capsule (IPACK) block in the reduction of postoperative pain, the need for rescue analgesics, and the effects on a range of motion (ROM) in patients with TKA. METHODS: This prospective randomized controlled study was carried out between February and May 2023. Based on the block method, 60 participants were divided into three equal groups. These groups included the IPACK block group (n=20), the genicular block group (n=20), and control group (n=20). Western Ontario and McMaster Universities Arthritis Index (WOMAC), Knee Society score (KSS) and Oxford Knee score (OKS) were used for clinical evaluation in the postoperative period. RESULTS: The KSS and OKS scores of the IPACK and GNB were significantly lower than the control group (p<0.001, p<0.001). The timed up and go (TUG) values of the IPACK and GNB groups at 12th and 24th hour were significantly lower than the control group (p<0.001, p<0.001). The Tramadol rescue values of the IPACK block and control groups were significantly higher than the GNB group (p=0.028, p=0.001, respectively). The ROM values of the IPACK and GNB groups were significantly higher than the control group (p<0.001, p<0.001). CONCLUSION: Both GNB and IPACK blocks had a significant positive impact on postoperative pain scores within the initial 24 hours following total knee arthroplasty (TKA). In comparison with IPACK, GNB had lower opioid consumption in the early postoperative period while also promoting better mobilization.

Characterization of the secretome of human tooth germ stem cells (hTGSCs) reveals neuro-protection by fine-tuning micro-environment.

Bone-marrow-derived mesenchymal stem cells (MSCs) demonstrate neuro-protective effects in several disease models. By producing growth-factors, cytokines and chemokines, they promote survival of neurons in damaged brain areas. Alternative MSC sources, such as human tooth germ stem cells (hTGSCs), have been investigated for their neuro-protective properties. They ameliorate effects of neuro-toxic agents by paracrine mechanisms, however these secreted bio-active molecules are not yet characterized. Therefore, the current study aimed to provide a detailed analysis of the secretome of hTGSCs. Brain cells were exposed to various toxic materials, including Alzheimer's ß-amyloid peptide (ß-AP) and 6-hydroxy-dopamine (6-OHDA). When co-cultured with hTGSCs, the activity of a number of anti-oxidant enzymes (catalase, glutathione-s-transferase, glutathione-peroxidase, superoxide-dismutase) was increased and neuronal death/apoptosis was subsequently reduced. The composition of the secreted bio-active materials is influenced by various pre-existing factors such as oxygen and glucose deprivation and the age of cells (passage number). This report reveals for the first time that the neuro-protective secretome of hTGSCs and the micro-environment of cells have a mutual and dynamic impact on one another.

Platelet Inventory and Using Out-of-group Platelet Suspension: A Cost-Effective Strategy for a Blood Transfusion Service.

BACKGROUND: Platelet (PLT) transfusions are essential for advanced hospitals, especially those with onco-hematology departments. However, platelet concentrates (PCs) have supply limitations and a shorter shelf life, which create difficulties for blood transfusion services (TSs). MATERIALS AND METHODS: This retrospective study was conducted over a 4-year period between January 2017 and January 2021 in a tertiary referral hospital. From the beginning of 2020, as a new strategy of our TS, a PLT inventory was produced and ABO-identical transfusions were prioritized when the inventory allowed; when this was not possible, ABO and Rh incompatible transfusion was employed. The numbers of transfused and discarded PCs were compared for each year. RESULTS: In 2017, a total of 799 PPCs were used and 70 PPCs were discarded with the expiration ratio (ER) of 8.0%. In 2018, 1124 PPCs were used and 99 PPCs were discarded with the ER of 7.4%. In 2019, 726 PPCs were used and 91 PPCs were discarded with the ER of 11.1%. In 2020, 1100 PPCs were used for 569 patients, of which 251 PPCs were ABO and Rh incompatible without any severe transfusion reaction. A total of 56 PPCs were discarded with the ER of 4.8%. CONCLUSION: The results of the current study suggested that with the determination of the platelet stock level and the use of out-of-group PCs, the rate of discarded PLT could be reduced. Nevertheless, based on current literature and experience, each TSs should make their own strategies and policies to provide an adequate supply of PCs.

Neurofilament Levels in CSF and Serum in an Adult SMA Cohort Treated with Nusinersen.

OBJECTIVE: To retrospectively evaluate the utility of serum and cerebrospinal fluid (CSF) levels of neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) as biomarkers for spinal muscular atrophy (SMA) progression and response to nusinersen treatment. METHODS: NfL and pNfH levels were quantified using single molecular array (SIMOA) in CSF of 33 adult SMA patients (SMN copy number 3-5) before and in response to nusinersen treatment. In 11 of the patients, blood serum samples were also collected. CSF NfL and pNfH from patients were compared to CSF Nfs from age-matched controls without neurological disease (n = 6). For patients, pearson correlation coefficients (r) were calculated to investigate associations between Nf levels and other functional outcome measures. RESULTS: Nf levels were similar between SMA and control adults and showed no change in response to nusinersen treatment in CSF or serum. Cross-sectional analyses showed an increase in CSF NfL and pNfH with age in patients (NfL p = 0.0013; pNfH p = 0.0035) and an increase in CSF NfL in controls (p = 0.002). In non-ambulatory patients, baseline serum pNfH showed a negative correlation with multiple strength and functional assessment metrics including Revised Upper Limb Module (r = -0.822, p = 0.04), upper extremity strength (r = -0.828, p = 0.042), lower extremity strength (r = -0.860, p = 0.028), and total strength (r = -0.870, p = 0.024). CONCLUSIONS: Nf levels did not change in response to nusinersen in adults with SMA and were not different from controls. In patients and controls, we detected an age-related increase in baseline CSF NfL and pNfH levels. Though some associations were identified, our results suggest Nf levels are not preditive or prognostic biomarkers in this population.

Differentiation and neuro-protective properties of immortalized human tooth germ stem cells.

Stem cells are considered to be promising therapeutic options in many neuro-degenerative diseases and injuries to the central nervous system, including brain ischemia and spinal cord trauma. Apart from the gold standard embryonic and mesenchymal origin, human tooth germ stem cells (hTGSCs) have also been shown to enjoy the characteristics of mesenchymal stem cells (MSCs) and the ability to differentiate into adipo-, chondro-, osteo- and neuro-genic cells, suggesting that they might serve as potential alternatives in the cellular therapy of various maladies. Immortalization of stem cells may be useful to avoid senescence of stem cells and to increase their proliferation potential without altering their natural characteristics. This study evaluated the expression of stem cell markers, surface antigens, differentiation capacity, and karyotype of hTGSCs that have been immortalized by human telomerase reverse transcriptase (hTERT) or simian vacuolating virus 40 (SV40) large T antigen. These undying cells were also evaluated for their neuro-protective potential using an in vitro SH-SY5Y neuro-blastoma model treated with hydrogen-peroxide or doxo-rubicin. Although hTGSC-SV40 showed abnormal karyotypes, our results suggest that hTGSC-hTERT preserve their MSC characteristics, differentiation capacity and normal karyotype, and they also possess high proliferation rate and neuro-protective effects even at great passage numbers. These peculiars indicate that hTGSC-hTERT could be used as a viable model for studying adipo-, osteo-, odonto- and neuro-genesis, as well as neuro-protection of MSCs, which may serve as a springboard for potentially utilizing dental waste material in cellular therapy.

Body weight reducing effect of oral boric acid intake.

BACKGROUND: Boric acid is widely used in biology, but its body weight reducing effect is not researched. METHODS: Twenty mice were divided into two equal groups. Control group mice drank standard tap water, but study group mice drank 0.28mg/250ml boric acid added tap water over five days. Total body weight changes, major organ histopathology, blood biochemistry, urine and feces analyses were compared. RESULTS: Study group mice lost body weight mean 28.1% but in control group no weight loss and also weight gained mean 0.09% (p<0.001). Total drinking water and urine outputs were not statistically different. Cholesterol, LDL, AST, ALT, LDH, amylase and urobilinogen levels were statistically significantly high in the study group. Other variables were not statistically different. No histopathologic differences were detected in evaluations of all resected major organs. CONCLUSION: Low dose oral boric acid intake cause serious body weight reduction. Blood and urine analyses support high glucose, lipid and middle protein catabolisms, but the mechanism is unclear.

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome.

Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1.

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Comparison and optimisation of transfection of human dental follicle cells, a novel source of stem cells, with different chemical methods and electro-poration.

INTRODUCTION: Human dental follicle cells (HDFCs) derived from human impacted third molars (wisdom teeth) have been shown to be a significant source of adult stem cells. Generation of mesenchymal stem cell-like cells from dental follicles causes minimal surgical stress. In vitro and in vivo reports showed that HDFCs can be utilized in gene and cell therapy applications which make them an attractive alternative source for different gene-cell therapy applications. However, there are currently no systematic comparative studies on transfection potential of HDFC cells using different chemical and electro-poration techniques. METHODS: Stem cells from impacted third tooth molars were isolated, and analyzed for expression of surface markers. Transfection efficiencies of four commercially available transfection reagents (Transfast, Escort V, Superfect and FuGene HD) and electro-poration on isolated stem cells were compared. RESULTS: Isolated HDFCs were stained positive for CD105, CD90, CD73, CD166, and negative for CD34, CD45, and CD133. Among the chemical transfection reagents used in this study, FuGene HD was the most efficient in transfecting HDFCs, even in the presence of 10% serum. CONCLUSION: Electro-poration of HDFCs yield relatively high transfection rates and cell viability when compared to chemical transfection techniques. Our observations might be useful for developing gene and cell therapy applications using dental follicle stem cells.

PUMILIO hyperactivity drives premature aging of Norad-deficient mice.

Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here, we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.

Efficacy of exogenous pyruvate in TremblerJ mouse model of Charcot-Marie-Tooth neuropathy.

INTRODUCTION: Classic Charcot-Marie-Tooth (CMT) neuropathies including those with Schwann cell genetic defects exhibit a length-dependent process affecting the distal axon. Energy deprivation in the distal axon has been the proposed mechanism accounting for length-dependent distal axonal degeneration. We hypothesized that pyruvate, an intermediate glycolytic product, could restore nerve function, supplying lost energy to the distal axon. METHODS: To test this possibility, we supplied pyruvate to the drinking water of the Trembler-J (TrJ ) mouse and assessed efficacy based on histology, electrophysiology, and functional outcomes. Pyruvate outcomes were compared with untreated TrJ controls alone or adeno-associated virus mediated NT-3 gene therapy (AAV1.NT-3)/pyruvate combinatorial approach. RESULTS: Pyruvate supplementation resulted increased myelinated fiber (MF) densities and myelin thickness in sciatic nerves. Combining pyruvate with proven efficacy from AAV1.tMCK.NT-3 gene therapy provided additional benefits showing improved compound muscle action potential amplitudes and nerve conduction velocities compared to pyruvate alone cohort. The end point motor performance of both the pyruvate and the combinatorial therapy cohorts was better than untreated TrJ controls. In a unilateral sciatic nerve crush paradigm, pyruvate supplementation improved myelin-based outcomes in both regenerating and the contralateral uncrushed nerves. CONCLUSIONS: This proof of principle study demonstrates that exogenous pyruvate alone or as adjunct therapy in TrJ may have clinical implications and is a candidate therapy for CMT neuropathies without known treatment.

A novel p.T139M mutation in HSPB1 highlighting the phenotypic spectrum in a family.

INTRODUCTION: Mutations in the HSPB1 gene encoding the small heat shock protein B1 are associated with an autosomal dominant, axonal form of Charcot-Marie-Tooth disease 2F (CMT2F) and distal hereditary motor neuropathy. Recently, distal myopathy had been described in a patient carrying HSPB1 mutation adding to the complexity of phenotypes resulting from HSPB1 mutations. METHODS: Five patients in a family with concerns of hereditary neuropathy were included. Detailed clinical examinations, including assessments of motor and sensory function, and electrophysiological data were obtained. Genetic analysis was requested through a commercial laboratory. In vitro studies were carried out to assess the pathogenicity of the novel mutation found in this family studies. RESULTS: All patients carried a novel mutation, c.146 C>T (p.T139M), substitution in the α-crystallin domain of HSPB1 causing a clinical phenotype with hyperreflexia and intrafamilial variability, from muscle cramps as the only presenting symptom to a classic CMT phenotype. In vitro studies showed that cells expressing HSPB1-T139M displayed decreased cell viability with increased expression of apoptosis markers. Moreover, overexpression of the mutant, not the wild-type HSPB1, caused formation of congophilic aggregates. CONCLUSIONS: In vitro findings strongly support the pathogenicity of this novel mutation. We propose that Congo red histochemical stain may serve as a simple screening tool for investigating if the aggregates in mutant cells have misfolded ß-pleated sheet secondary structures.

Impaired regeneration in calpain-3 null muscle is associated with perturbations in mTORC1 signaling and defective mitochondrial biogenesis.

BACKGROUND: Previous studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle. METHODS: To gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-ß and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle. RESULTS: At 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-ß transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers. CONCLUSION: Our findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy.

Tooth replantation with adipose tissue stem cells and fibrin sealant: microscopic analysis of rat's teeth.

Treatment for dental avulsion cases is early or late replantation of the traumatized teeth. Prognosis of the replanted tooth depends on the level of periodontal injury. Adipose tissue stem cells (ATSCs) were reported to improve periodontal ligament tissue (PDL) regeneration. Fibrin sealant (FS) contains thrombin and fibrinogen to form an adhesive fibrin clot routinely used in surgical procedures. Here, we aimed to investigate the effects of ATSCs + FS treatment on healing of PDL after tooth replantation in a rat model. After 60 min of extraction, maxillary central incisor teeth were replanted with ATSCs + FS. Two months later, the rats were sacrificed and hemimaxilla blocks were dissected out for histological analysis. The results showed that there was a significant improvement in histological findings of ATSCs + FS treated group compared to only FS treated and non-treated groups corresponding to reduced inflammatory resorption and increased new PDL formation. Furthermore, the ankylosis levels were lowered after ATSCs + FS treatment. Singular use of FS improved PDL healing moderately. Our results indicated that ATSCs + FS treatment improves PDL healing after tooth replantation suggesting a new therapeutic potential in the treatment of dental avulsion cases.

Bone marrow mesenchymal stem cells enhance bone formation in orthodontically expanded maxillae in rats.

OBJECTIVE: To transplant bone marrow-derived mesenchymal stem cells (MSCs) into the interpremaxillary suture after rapid maxillary expansion with the aim of increasing new bone formation in the suture. MATERIALS AND METHODS: Nineteen male Wistar rats were divided into two groups (control, n  =  9; experimental, n  =  10). Both groups were subjected to expansion for 5 days, and 50 cN of force was applied to the maxillary incisors with a helical spring. Pkh67(+) (green fluorescent dye)-labeled MSCs were applied to the interpremaxillary suture after force application into the interpremaxillary suture of rats. Bone formation in the sutural area was histomorphometrically evaluated, including the amount of new bone formation (µm(2)), number of osteoblasts, number of osteoclasts, and number of vessels. Mann-Whitney U-test was used for statistical evaluation at the P < .05 level. RESULTS: After 10 days of retention, Pkh67(+) can be detected in suture mostly in the injection site under fluorescence microscope. Histomorphometric analysis revealed that a single local injection of MSCs into the midpalatal suture increased the new bone formation in the suture by increasing the number of osteoblasts and new vessel formation, compared with controls injected with phosphate-buffered saline. CONCLUSIONS: This preclinical study might provide foundations for the underlying potential clinical use of MSCs after maxillary expansion. Given the fact that MSCs are currently in use in clinical trials, this approach might be a feasible treatment strategy to accelerate new bone tissue formation in midpalatal suture and to shorten the treatment period for patients undergoing maxillary expansion reinforcement.

Bmp 2 and bmp 7 induce odonto- and osteogenesis of human tooth germ stem cells.

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.