Acute diabetic foot in post kidney transplantation patients receiving chronic immunosuppression-clinical presentation and outcomes.

AIMS: Data regarding diabetic foot ulcers in patients after solid organ transplantation, particularly kidney transplantation, are limited. Chronic immunosuppression may be associated with impaired wound healing and a higher risk of amputations. In this study, we characterised the clinical presentation and outcomes of patients after kidney transplantation admitted to the diabetic foot unit, compared to non-kidney-transplant patients. MATERIALS AND METHODS: Data on the baseline characteristics, clinical presentation, and outcomes of all patients admitted to the diabetic foot unit of a large tertiary centre between the years 2014 and 2019 were collected. The most recent admission of each patient was considered. Primary outcomes were major amputations and 1 year mortality rate. RESULTS: During the study period, 537 patients were hospitalised, 18 of them were receiving immunosuppressive therapy due to kidney transplantation. Baseline characteristics of the patients were broadly similar, except that smoking was reported by 22.0% of the non-transplant patients and by none of the post-transplant patients (p = 0.01). Post-transplant patients tended to be younger (59.4 ± 11.1 vs. 65.3 ± 12.2; p = 0.07), were more likely to have type-1 diabetes (16.7% vs. 5.2%; p = 0.07) and had lower glucose levels upon admission (9.4 ± 4.3 vs. 12.0 ± 6.4 mmol/L; p = 0.07). Overall, 30% of the patients underwent major amputation, in-patient mortality rate was 9.3%, and 1 year mortality rate was 27.2%. Rates were similar in the post-transplant versus the non-post-transplant patients (p = 0.83, 1.00, 0.59, respectively). CONCLUSIONS: Post-transplant patients did not incur worse outcomes in spite of immunosuppressive therapy. Limb salvage efforts should be pursued in these patients similar to the overall population.

Treatment of diabetic foot ulcers in a frail population with severe co-morbidities using at-home photobiomodulation laser therapy: a double-blind, randomized, sham-controlled pilot clinical study.

PURPOSE: To evaluate the safety and efficacy of an at-home photobiomodulation (PBM) device for the treatment of diabetic foot ulcers (DFUs) in a frail population with severe comorbidities. METHODS: Prospective, randomized, double-blind, sham-controlled pilot study. Patients (age = 63 ± 11 years, male:female 13:7) with insulin-dependent diabetes type 2, neuropathy, peripheral artery disease, significant co-morbidities, and large osteomyelitis-associated DFUs (University of Texas grade ≥ III) were randomized to receive active (n = 10) or sham (n = 10) at-home daily PBM treatments (pulsed near-infrared 808 nm Ga-Al-As laser, 250 mW, 8.8 J/cm2) for up to 12 weeks in addition to standard care. The primary outcome was the %wound size reduction. The secondary was adverse events. RESULTS: With the numbers available, PBM-treated group had significantly greater %reduction compared to sham (area [cm2], baseline vs endpoint: PBM 10[20.3] cm2 vs 0.2[2.4] cm2; sham, 7.9 [12.0] cm2 vs 4.6 [13.8] cm2, p = 0.018 by Mann-Whitney U test). Wound closure > 90% occurred in 7 of 10 PBM-treated patients but in only 1 of 10 sham patients (p = 0.006). No adverse device effects were observed. CONCLUSIONS: Photobiomodulation at home, in addition to standard care, may be effective for the treatment of severe DFUs in frail patients with co-morbidities and is particularly relevant at these times of social distancing. Our preliminary results justify the conduction of a larger clinical trial. CLINICALTRIALS: gov: NCT01493895.

[HOSPITALIZATIONS DUE TO ACUTE DIABETIC FOOT: ANNUAL TRENDS AND PREDICTORS OF MORBIDITY AND MORTALITY - 5-YEARS EXPERIENCE OF A MULTIDISCIPLINARY UNIT].

INTRODUCTION: Since 2012, patients presenting to our hospital with an acute diabetic foot are hospitalized in a dedicated unit. This study describes patients' characteristics and trends in amputations, procedures and mortality during the years 2014-2018. METHODS: We retrospectively reviewed the electronic medical records of 694 patients admitted to the unit during the study period. We collected demographic, clinical and laboratory data, procedures and outcomes. Annual trends were studied as well as predictors to any or major amputation and to mortality within 1 year following discharge. RESULTS: The mean age was 63.8±12.7 years and 75.4% of the patients were male. There was a high prevalence of neuropathy, peripheral artery disease and ischemic heart disease (55.3%, 66.1% and 44.2% respectively). Previous hospitalization was noted for 62.0% of the patients and 38.3% had undergone a previous amputation. The majority of the patients had chronic kidney disease and 19.0% were dialysis patients. During hospitalization, 54.3% of the patients underwent any amputation, 25.2% had a major amputation and 6.2% died. The mortality rate within 1 year of discharge was 24.5%. There were no changes in patient demographics, characteristics or outcomes during the study years, although an increase in the proportion of patients who had undergone previous amputation, and of current smokers in recent years was noted. Moreover, in recent years more vascular procedures and surgical procedures in the operating room were performed. Older age, recent hospitalization, previous amputation, neuropathy, ischemic heart disease, peripheral vascular disease, chronic renal insufficiency, elevated inflammatory markers, a progressive ulcer, and a midfoot or hindfoot (vs. forefoot) ulcer were all associated with major amputations. CONCLUSIONS: During the study period, patients' characteristics remained generally stable as did amputation and mortality rates. The high 1-year mortality rate of this population is indicative of these patients' significant morbidity.

Serum NT/CT SIRT1 ratio reflects early osteoarthritis and chondrosenescence.

OBJECTIVE: Previous work has established that the deacetylase sirtuin-1 (SIRT1) is cleaved by cathepsin B in chondrocytes subjected to proinflammatory stress, yielding a stable but inactive N-terminal (NT) polypeptide (75SIRT1) and a C-terminal (CT) fragment. The present work examined if chondrocyte-derived NT-SIRT1 is detected in serum and may serve as an investigative and exploratory biomarker of osteoarthritis (OA). METHODS: We developed a novel ELISA assay to measure the ratio of NT to CT of SIRT1 in the serum of human individuals and mice subjected to post-traumatic OA (PTOA) or age-dependent OA (ADOA). We additionally monitored NT/CT SIRT1 in mice subject to ADOA/PTOA followed by senolytic clearance. Human chondrosenescent and non-senescent chondrocytes were exposed to cytokines and analysed for apoptosis and NT/CT SIRT1 ratio in conditioned medium. RESULTS: Wild-type mice with PTOA or ADOA of moderate severity exhibited increased serum NT/CT SIRT1 ratio. In contrast, this ratio remained low in cartilage-specific Sirt1 knockout mice despite similar or increased PTOA and ADOA severity. Local clearance of senescent chondrocytes from old mice with post-traumatic injury resulted in a lower NT/CT ratio and reduced OA severity. While primary chondrocytes exhibited NT/CT ratio increased in conditioned media after prolonged cytokine stimulation, this increase was not evident in cytokine-stimulated chondrosenescent cells. Finally, serum NT/CT ratio was elevated in humans with early-stage OA. CONCLUSIONS: Increased levels of serum NT/CT SIRT1 ratio correlated with moderate OA in both mice and humans, stemming at least in part from non-senescent chondrocyte apoptosis, possibly a result of prolonged inflammatory insult.

Tuftelin's involvement in embryonic development.

Little is known about tuftelin expression in the developing embryo, previously it was thought to play a role in tooth enamel mineralization. In this study we show tuftelin's spatio-temporal expression in mineralizing and nonmineralizing tissues of the craniofacial complex in the developing mouse embryo. Embryos aged E10.5-E18.5 and newborns aged P3 were used in this study. Polymerase chain reaction (PCR), Real-time PCR, sequencing, and in-situ hybridization were used to detect and quantify messenger RNA (mRNA) expression in different developmental stages. We applied indirect immunohistochemistry and western-blot analyses to investigate protein expression. Two tuftelin mRNA transcripts and a single 64KDa protein were detected throughout embryonic development. Tuftelin was detected in tissues which develop from different embryonic origins; ectoderm, ectomesenchyme, and mesoderm. Tuftelin mRNA and protein were expressed already at E10.5, before the initiation of tooth formation and earlier than previously described. The expression pattern of tuftelin mRNA and protein exhibits dynamic spatio-temporal changes in various tissues. Tuftelin is expressed in neuronal tissues, thus fitting with its described correlation to nerve growth factor. A shift between cytoplasmatic and perinuclear/nuclear expression implies a possible role in regulation of transcription. Recent studies showed tuftelin is induced under hypoxic conditions in-vitro and in-vivo, through the hypoxia-inducible factor 1-α pathway. These results led to the hypothesis that tuftelin is involved in adaptation to hypoxic conditions. The fact that much of mammalian embryogenesis occurs at O 2 concentrations of 1-5%, raises the possibility that tuftelin expression throughout development is due to its role in the adaptive mechanisms in response to hypoxia.

[FROM THE LAB BENCH TO THE PATIENT - CAN INJURED SKELETAL TISSUES REGENERATE?]

INTRODUCTION: Regenerative medicine research has evolved significantly in recent years. There is a great un-met clinical need for developing new treatments that will induce regeneration of injured skeletal tissues in cases such as large bony defects caused by trauma or tumor resection, articular cartilage defects and torn or degenerate tendons and ligaments. Except for bone that can regenerate small defects, all other skeletal tissues do not hold the natural capability for regeneration after injury and rather form a less functional scar tissue. In order to induce tissue regeneration, it is now believed that three crucial elements must reach the injured zone: a) multipotent cells that can rapidly proliferate and differentiate to form the injured tissues, such as mesenchymal stem cells for skeletal tissues; b) extra-cellular matrix that will support the newly built tissues, and c) the correct molecular signals. Using diverse research tools and expertise, our department focused its research on basic, translational and clinical solutions for injured and degenerative skeletal tissues. In this review we will describe our different research directions, from in-vitro cell cultures and animal models studies to human clinical trials.

Skeletal ligament healing using the recombinant human amelogenin protein.

Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM(+)) resulted in enhanced healing of the tooth-supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM(+), dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 µg/µl rHAM(+) were similar to the normal un-transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 µg/µl rHAM(+) treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 µg/µl rHAM(+) increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM(+) dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers.

Real-time Reconstruction of Comminuted Mandibular Fractures Using 3D Printing.

Background: Comminuted fractures of the jaws are complex injuries requiring special attention. In the past, treatment included closed reduction using maxillomandibular fixation. With advancements in technology and fixation systems, open reduction became a prevalent option. These fractures are difficult to reconstruct during the primary treatment phase, thus resulting in higher complication rates. The introduction of three-dimensional (3D) planning and printing brought about superior outcomes, yet these focus on secondary reconstruction due to the need for outsourcing planning and titanium printing. Methods: In this report, we describe real-time in-house 3D planning and printing using computer-assisted design software and a 3D-fused deposition printer for virtual reduction of the comminuted fractures and printing of the reconstructed mandible. Results: Following virtual 3D reduction, the newly created mandibles were 3D printed. The model was then used to preband a reconstruction plate, which in turn was used as a template during surgery for reducing the segments. The process of virtual reduction and printing should take a couple of hours at most. The results of five cases showed good alignment and proper function. Conclusion: Three-dimensional technology can be applied in the everyday primary care treatment protocol of comminuted fractures as an in-house tool which greatly improves both functional and aesthetic outcomes.

75-kd sirtuin 1 blocks tumor necrosis factor α-mediated apoptosis in human osteoarthritic chondrocytes.

OBJECTIVE: Sirtuin 1 (SirT1) has been implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic (OA) chondrocytes to tumor necrosis factor α (TNFα) generates a stable and enzymatically inactive 75-kd form of SirT1 (75SirT1) via cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we hypothesized that it has a distinct role in OA, and the present study was undertaken to identify this role. METHODS: The presence of cathepsin B and 75SirT in OA and normal human chondrocytes was analyzed. Confocal imaging of SirT1 was used to monitor its subcellular trafficking following TNFα stimulation. Coimmunofluorescence staining for cathepsin B, mitochondrial cytochrome oxidase subunit IV, and lysosome-associated membrane protein 1 together with SirT1 was performed. Human chondrocytes were tested for apoptosis by fluorescence-activated cell sorter analysis and immunoblotting for caspases 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1-cytochrome c association. RESULTS: Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistent with this, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with cytochrome c following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of full-length SirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα. Levels of cathepsin B and 75SirT1 were elevated in OA versus normal chondrocytes. Additional analyses showed that human chondrocytes exposed to OA-derived synovial fluid generated the 75SirT1 fragment. CONCLUSION: These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.

Enterococci in Diabetic Foot Infections: Prevalence, Clinical Characteristics, and Outcomes.

Background: Diabetic foot infections (DFIs) are frequently polymicrobial, yet the relevance of each isolated pathogen, remains ill-defined. Specifically, the prevalence and pathogenicity of enterococcal DFIs and the impact of targeted antienterococcal treatment remain elusive. Methods: We collected demographic, clinical, and outcome-related data on patients admitted with DFIs to the Hadassah Medical Center diabetic foot unit between 2014 and 2019. The primary outcome was a composite of in-hospital death or major amputation. Secondary outcomes included any amputation, major amputation, length of stay (LOS), and 1-year major amputation or mortality rate. Results: Enterococci were isolated in 35% of 537 eligible DFI case patients, who were notable for a higher prevalence of peripheral vascular disease, increased levels of C-reactive protein, and higher Wagner scores. Infection in enterococci-positive individuals was mostly polymicrobial (96.8% vs 61.0% in non-enterococci-infected patients; P < .001). Enterococci-infected patients were more likely to undergo amputation (72.3% vs 50.1%; P < .001) and had longer hospital stays (median LOS, 22.5 vs 17 days; P < .001), but the primary end point of major amputation or in-hospital death did not differ between groups (25.5% vs 21.0%; P = .26). Appropriate antienterococcal antibiotics were used in 78.1% of enterococci-infected patients and, compared with results in untreated patients, were associated with a trend toward a lower rate of major amputations (20.4% vs 34.1%; P = .06) but longer hospitalization (median LOS, 24 vs 18 days; P = .07). Conclusions: Enterococci are common in DFIs and associated with higher rates of amputation and longer hospitalization. A reduction in major amputation rates with appropriate enterococci treatment is suggested retrospectively, meriting validation by future prospective studies.

Regeneration of injured articular cartilage using the recombinant human amelogenin protein.

Aims: Cartilage injuries rarely heal spontaneously and often require surgical intervention, leading to the formation of biomechanically inferior fibrous tissue. This study aimed to evaluate the possible effect of amelogenin on the healing process of a large osteochondral injury (OCI) in a rat model. Methods: A reproducible large OCI was created in the right leg femoral trochlea of 93 rats. The OCIs were treated with 0.1, 0.5, 1.0, 2.5, or 5.0 µg/µl recombinant human amelogenin protein (rHAM+) dissolved in propylene glycol alginate (PGA) carrier, or with PGA carrier alone. The degree of healing was evaluated 12 weeks after treatment by morphometric analysis and histological evaluation. Cell recruitment to the site of injury as well as the origin of the migrating cells were assessed four days after treatment with 0.5 µg/µl rHAM+ using immunohistochemistry and immunofluorescence. Results: A total of 12 weeks after treatment, 0.5 µg/µl rHAM+ brought about significant repair of the subchondral bone and cartilage. Increased expression of proteoglycan and type II collagen and decreased expression of type I collagen were revealed at the surface of the defect, and an elevated level of type X collagen at the newly developed tide mark region. Conversely, the control group showed osteoarthritic alterations. Recruitment of cells expressing the mesenchymal stem cell (MSC) markers CD105 and STRO-1, from adjacent bone marrow toward the OCI, was noted four days after treatment. Conclusion: We found that 0.5 µg/µl rHAM+ induced in vivo healing of injured articular cartilage and subchondral bone in a rat model, preventing the destructive post-traumatic osteoarthritic changes seen in control OCIs, through paracrine recruitment of cells a few days after treatment.

The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation.

The tuftelin protein isoforms undergo post-translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down-regulation of HIF1α mRNA blocked hypoxia-induced HIF1α expression, and reduced by 89% hypoxia-induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF-mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA-signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the "hypoxic genome," and NGF-induced neurotrophic and angiogenic effects.

Pyogenic flexor tenosynovitis in children.

Pyogenic flexor tenosynovitis is an uncommon, emergent hand infection. The literature lacks any description of the disease and the variability of its manifestations in young children. We describe 3 cases. Two cases were diagnosed and treated promptly, and the third presented late, with atypical clinical signs, causing a delay in his diagnosis and treatment and stressing the caution to be taken with the evaluation of these children with signs of hand infection.

Angiogenic potential of mesenchymal stem cells derived from patients with diabetes seeded on decellularized micro fragments.

Mesenchymal stem cells (MSCs) are a potential source of angiogenic factors which may promote wound healing in poorly vascularized diabetic foot ulcers. We demonstrate that MSCs of patients with diabetic foot ulcers seeded on decellularized micro-fragments transcribe and secrete angiogenic factors in amounts comparable to MSCs derived from healthy individuals.

Predictors and outcomes of diabetic foot ulcer infection with ESBL-producing bacteria in a large tertiary center.

OBJECTIVES: The aim of this study was to describe the predictors and outcomes of infection with extended-spectrum beta-lactamase (ESBL)-producing bacteria in patients with an acute diabetic foot infection (DFI). METHODS: The records of patients admitted with acute DFI to a large tertiary hospital during the years 2014-2018 were reviewed. Demographic, clinical, and laboratory data were collected, as well as outcomes regarding amputations and mortality. Only cultures obtained during the first 2 weeks following admission were considered. RESULTS: Cultures were available for 493 patients; 121 (24.5%) included bacteria suspected of being ESBL producers. Patients infected with ESBL-producing bacteria were older, more likely to have peripheral vascular disease (PVD), and had higher SINBAD and Wagner scores upon admission. They were also more likely to have been hospitalized in the recent 6 months. Major amputations were more prevalent in patients with versus without an ESBL-producing bacterial infection (30.6% vs 19.4%; P = 0.010), yet overall amputations and mortality rates were similar. CONCLUSIONS: ESBL-producing bacteria are common pathogens in DFI, more prevalent in older patients with PVD, advanced ulcers, and recent hospitalization. They are associated with higher rates of major amputation. These considerations may support the choice of empirical antibiotic therapy in patients admitted with an acute DFI.

Regeneration of grade 3 ankle sprain, using the recombinant human amelogenin protein (rHAM+ ) in a rat model.

Lateral ligament tears, also known as high-grade ankle sprains, are common, debilitating, and usually heal slowly. Ten to thirty percent of patients continue to suffer from chronic pain and ankle instability even after 3 to 9 months. Previously, we showed that the recombinant human amelogenin (rHAM+ ) induced regeneration of fully transected rat medial collateral ligament, a common proof-of-concept model. Our aim was to evaluate whether rHAM+ can regenerate torn ankle calcaneofibular ligament (CFL), an important component of the lateral ankle stabilizers. Right CFLs of Sabra rats were transected and treated with 0, 0.5, or 1 µg/µL rHAM+ dissolved in propylene glycol alginate (PGA). Results were compared with the normal group, without surgery. Healing was evaluated 12 weeks after treatment by mechanical testing (ratio between the right and left, untransected ligaments of the same rat), and histology including immunohistochemical staining of collagen I and S100. The mechanical properties, structure, and composition of transected ligaments treated with 0.5 µg/µL rHAM+ (experimental) were similar to untransected ligaments. PGA (control) treated ligaments were much weaker, lax, and unorganized compared with untransected ligaments. Treatment with 1 µg/µL rHAM+ was not as efficient as 0.5 µg/µL rHAM+ . Normal arrangement of collagen I fibers and of proprioceptive nerve endings, parallel to the direction of the force, was detected in ligaments treated with 0.5 µg/µL rHAM+ , and scattered arrangement, resembling scar tissue, in control ligaments. In conclusion, we showed that rHAM+ induced significant mechanical and structural regeneration of torn rat CFLs, which might be translated into treatment for grades 2 and 3 ankle sprain injuries.

Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis.

Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth-supporting tissues, alveolar bone, periodontal-ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM(+)), induced in vivo regeneration of all tooth-supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM(+) induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio-temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM(+).

Amelogenin in cranio-facial development: the tooth as a model to study the role of amelogenin during embryogenesis.

The amelogenins comprise 90% of the developing extracellular enamel matrix proteins and play a major role in the biomineralization and structural organization of enamel. Amelogenins were also detected, in smaller amounts, in postnatal calcifying mesenchymal tissues, and in several nonmineralizing tissues including brain. Low molecular mass amelogenin isoforms were suggested to have signaling activity; to produce ectopically chondrogenic and osteogenic-like tissue and to affect mouse tooth germ differentiation in vitro. Recently, some amelogenin isoforms were found to bind to the cell surface receptors; LAMP-1, LAMP-2 and CD63, and subsequently localize to the perinuclear region of the cell. The recombinant amelogenin protein (rHAM(+)) alone brought about regeneration of the tooth supporting tissues: cementum, periodontal ligament and alveolar bone, in the dog model, through recruitment of progenitor cells and mesenchymal stem cells. We show that amelogenin is expressed in various tissues of the developing mouse embryonic cranio-facial complex such as brain, eye, ganglia, peripheral nerve trunks, cartilage and bone, and is already expressed at E10.5 in the brain and eye, long before the initiation of tooth formation. Amelogenin protein expression was detected in the tooth germ (dental lamina) already at E13.5, much earlier than previously reported (E19). Application of amelogenin (rHAM(+)) beads together with DiI, on E13.5 and E14.5 embryonic mandibular mesenchyme and on embryonic tooth germ, revealed recruitment of mesenchymal cells. The present results indicate that amelogenin has an important role in many tissues of the cranio-facial complex during mouse embryonic development and differentiation, and might be a multifunctional protein.

Association of Inpatient Glucose Measurements With Amputations in Patients Hospitalized With Acute Diabetic Foot.

CONTEXT: The association of inpatient glucose measurements with amputations in patients admitted with acute diabetic foot has not been described. OBJECTIVE: To evaluate the relationship of hyperglycemia, hypoglycemia, and glucose variability during hospitalization with amputations in patients hospitalized with acute diabetic foot. DESIGN: Retrospective cohort study. SETTING: Academic tertiary hospital. PATIENTS: We reviewed demographic, clinical, laboratory, and point-of-care glucose data in patients hospitalized with acute diabetic foot in the Diabetic Foot Unit during 2015 through 2017. MAIN OUTCOME MEASURES: The primary outcomes were any or major amputations during hospitalization. Secondary outcomes included length of hospitalization and in-hospital mortality. RESULTS: During the study period, 418 patients were hospitalized in the Diabetic Foot Unit and 45,496 glucose measurements were taken. Patients experiencing any hyperglycemia and any or severe hypoglycemia were more likely to undergo any or major amputations during hospitalization. High glycemic variability was associated with major amputations. Peripheral vascular disease (PVD), high Wagner score, and hypoglycemia were independent predictors of amputations. Older age, PVD, previous amputation, elevated white blood cell level, high Wagner score, and hypoglycemia were independent predictors of major amputations. CONCLUSIONS: In-patient hypoglycemia emerged as an independent risk factor for any and major amputations. Although it is unclear whether hypoglycemia directly contributes to adverse outcomes or is simply a biomarker of disease severity, efforts to minimize in-hospital hypoglycemic events are warranted.

Tuftelin Is Required for NGF-Induced Differentiation of PC12 Cells.

Nerve growth factor (NGF) promotes pleiotropic gene transcription-dependent biological effects, in neuronal and non-neuronal cells, including survival, proliferation, differentiation, neuroprotection, pain, and angiogenesis. It is hypothesized that during odontogenesis, NGF may be implicated in morphogenetic and mineralization events by affecting proliferation and/or differentiation of dental cells. Tuftelin belongs to the enamel associated teeth proteins and is thought to play a role in enamel mineralization. We previously reported that tuftelin transcript and protein, which are ubiquitously expressed in various tissues of embryos, adults, and tumors, were significantly upregulated during NGF-induced PC12 differentiation. To further confirm the involvement of tuftelin in the differentiation process, we established a tuftelin-knockdown neuronal PC12 cell model, using a non-cytotoxic siRNA directed towards sequences at the 3' UTR of the tuftelin gene. Using real-time PCR, we quantified tuftelin mRNA expression and found that tuftelin siRNA, but not scrambled siRNA or transfection reagents, efficiently depleted about 60% of NGF-induced tuftelin mRNA transcripts. The effect of tuftelin siRNA was quantified up to 6 days of NGF-induced differentiation. Using immunofluorescence and western blot analyses, we also found a direct correlation between reduction of 60-80% in tuftelin protein expression and inhibition of about 50-70% in NGF-induced differentiation of the cells, as was detected after 3-6 days of treatment. These results demonstrate an important role for tuftelin in NGF-induced differentiation of PC12 cells. Tuftelin could be a useful target for drug development in disease where neurotrophin therapy is required.