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.

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.

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.

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.

[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.

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.

Outcome of femoral fractures in poliomyelitis patients.

BACKGROUND AND PURPOSE: As patients who were afflicted with poliomyelitis during the outbreaks in the past are aging, lower extremity osteoporotic fractures are becoming more frequent. Fixation in deformed, porotic bone, coupled with muscle weakness and imbalance creates a unique challenge when treating these fractures as does their reduced rehabilitation potential. The aim of this study was to investigate the outcome of femoral fractures in surviving poliomyelitis patients. PATIENTS AND METHODS: Sixty-five patients with 74 femoral fractures were treated between 1990 and 2014. Clinical outcome was assessed using the Parkland and Palmer mobility score, and quality-of-life was assessed using the SF-12® score. RESULTS: Some 84% of the fractures were a result of low-energy mechanisms and occurred in the polio-affected limbs, but nonaffected limbs were also injured owing to low-energy mechanisms in all cases. Fifty-seven fractures were treated operatively. There were nine re-operations (16%), including implant removals, nonunion, peri-implant fractures, and malunion. Some 60% of the patients did not regain their previous ambulatory capacity. Post-operative weight-bearing status did not correlate with the final functional outcome. CONCLUSIONS: Polio patients with femoral fractures have a guarded prognosis for regaining their pre-injury ambulatory capacity. A higher re-operation rate than that with "normal" osteoporotic fractures is expected.

Closed Humeral Fracture Complicated with Acute Hematogenous Osteomyelitis: A Case Report.

INTRODUCTION: Acute hematogenous osteomyelitis (AHO) has been noted mainly in open fractures injuring soft tissue immunological defenses and in immuneincompetent patients. Osteomyelitis complicating closed fractures in immunocompetent adult patients is, therefore, a rare clinical entity with scarce literature. CASE REPORT: We report a case of primary Staphylococcus aureus bacterial infection of a closed, humeral shaft fracture occurring in a previously healthy 28-year-old male patient. The patient was involved in a motorcycle accident and was admitted to the surgical ward with a chest drain. While hospitalized, a peak of fever was noted, but no source was found. Diagnosis of the closed fracture infection was noted on primary open reduction and internal fixation (ORIF), and although the patient was treated with antibiotics, local osteomyelitis developed. Treatment including serial debridements utilizing gentamycin beads and an additional ORIF procedure until the full union was achieved. The patient regained full, painless, motion of the arm and shoulder. CONCLUSION: Although AHO complicating a closed fracture in immunocompetent adults is very rare, it should not be overlooked, and special attention should be sought in such cases. Meticulous debridement and rigid fixation are utmost for the eradication of infection and fracture union. Patients presenting with such infections should, therefore, be followed closely and treated promptly.

Anti-T lymphocyte globulin (ATG) induces generation of regulatory T cells, at least part of them express activated CD44.

We show here that the anti-T lymphocyte immunoglobulin (ATG) can induce Treg cells following 24-h incubation in human peripheral blood mononuclear cells (PBMCs). The ATG-induced Treg cells express known cell surface markers (e.g., CD25, FoxP3) and suppress the proliferation of autologous responder PBMCs, stimulated with allogeneic PBMCs, when added into the mixed lymphocyte culture (MLC) at zero time point or 48 h later. We expanded the characteristics of the ATG-induced human Treg cells by showing that they express a novel biomarker designated "activated CD44". ATG-induced Treg cells retain their suppressor function after freezing and thawing or irradiation. Suppression of MLC by ATG-induced Treg cells is consistently seen when the Treg cells and the responder cells were derived from the same donor, but not when they derived from different donors. Finally, patients undergoing stem cell transplantation and conditioned with ATG generate in vivo Treg cells that suppress MLC.

Regulatory T-cells in Graves' orbitopathy: baseline findings and immunomodulation by anti-T lymphocyte globulin.

BACKGROUND AND AIM: Graves' orbitopathy (GO) is characterized by orbital T cell infiltration and local release of proinflammatory cytokines. We aimed to evaluate the involvement of baseline regulatory T (Treg) cells and rabbit anti-T lymphocyte globulin (rATG)-induced Treg cells in GO. DESIGN: Peripheral blood mononuclear cells (PBMCs) from seven patients with Graves' disease (GD) without eye manifestations, 29 patients with GO, and 15 healthy controls were incubated with rATG, washed, and analyzed for expression of Treg cell markers and for ability to suppress mixed lymphocyte reaction. RESULTS: Elevation of CD4 to CD8 ratio and enhanced secretion of IL-6, IL-10, and TNFα were detected in PBMCs of GO patients compared with controls (both P < 0.01). Despite this abnormality, the frequencies of CD4(+)CD25(+)FoxP3(+) of GO and control PBMCs were similar and remained unchanged after 24 h incubation with control rabbit IgG (rIgG). Incubation with polyclonal rATG increased the frequency of PBMCs of GO patients, expressing Treg cell markers (CD25, FoxP3, and the IL-7 receptor CD127(low/-)) by 2.5-8 fold over corresponding rIgG-incubated cells (P < 0.05). FoxP3/CD4 rATG-induced Treg cell marker expressed more intensively on GO peripheral blood leukocytes (PBLs) than on GD (P < 0.01) or normal (P < 0.05) PBLs, yet its expression on normal PBLs was stronger than on GD PBLs (P < 0.05). GO rATG-incubated PBMCs, but not rIgG-incubated PBMCs, suppressed (P < 0.05) proliferation of autologous responder cells stimulated with allogeneic irradiated cells in mixed lymphocyte reaction. Such rATG-induced suppressive activity was not detected in GD. CONCLUSION: This study is the first to show that PBMCs of patients with GO substantially increase Treg cells in both frequency and potency after in vitro incubation with rATG.

MT1-MMP and RECK are involved in human CD34+ progenitor cell retention, egress, and mobilization.

The mechanisms governing hematopoietic progenitor cell mobilization are not fully understood. We report higher membrane type 1-MMP (MT1-MMP) and lower expression of the MT1-MMP inhibitor, reversion-inducing cysteine-rich protein with Kazal motifs (RECK), on isolated circulating human CD34+ progenitor cells compared with immature BM cells. The expression of MT1-MMP correlated with clinical mobilization of CD34+ cells in healthy donors and patients with lymphoid malignancies. Treatment with G-CSF further increased MT1-MMP and decreased RECK expression in human and murine hematopoietic cells in a PI3K/Akt-dependent manner, resulting in elevated MT1-MMP activity. Blocking MT1-MMP function by Abs or siRNAs impaired chemotaxis and homing of G-CSF-mobilized human CD34+ progenitors. The mobilization of immature and maturing human progenitors in chimeric NOD/SCID mice by G-CSF was inhibited by anti-MT1-MMP treatment, while RECK neutralization promoted motility and egress of BM CD34+ cells. BM c-kit+ cells from MT1-MMP-deficient mice also exhibited inferior chemotaxis, reduced homing and engraftment capacities, and impaired G-CSF-induced mobilization in murine chimeras. Membranal CD44 cleavage by MT1-MMP was enhanced following G-CSF treatment, reducing CD34+ cell adhesion. Accordingly, CD44-deficient mice had a higher frequency of circulating progenitors. Our results reveal that the motility, adhesion, homing, and mobilization of human hematopoietic progenitor cells are regulated in a cell-autonomous manner by dynamic and opposite changes in MT1-MMP and RECK expression.