The Influence of Cell Source and Donor Age on the Tenogenic Potential and Chemokine Secretion of Human Mesenchymal Stromal Cells.

BACKGROUND: Cellular therapy is proposed for tendinopathy treatment. Bone marrow- (BM-MSC) and adipose tissue- (ASC) derived mesenchymal stromal cells are candidate populations for such a therapy. The first aim of the study was to compare human BM-MSCs and ASCs for their basal expression of factors associated with tenogenesis as well as chemotaxis. The additional aim was to evaluate if the donor age influences these features. METHODS: Cells were isolated from 24 human donors, 8 for each group: hASC, hBM-MSC Y (age ≤ 45), and hBM-MSC A (age > 45). The microarray analysis was performed on RNA isolated from hASC and hBM-MSC A cells. Based on microarray results, 8 factors were chosen for further evaluation. Two genes were additionally included in the analysis: SCLERAXIS and PPARγ. All these 10 factors were tested for gene expression by the qRT-PCR method, and all except of RUNX2 were additionally evaluated for protein expression or secretion. RESULTS: Microarray analysis showed over 1,400 genes with a significantly different expression between hASC and hBM-MSC groups. Eight of these genes were selected for further analysis: CXCL6, CXCL12, CXCL16, TGF-ß2, SMAD3, COLLAGEN 14A1, MOHAWK, and RUNX2. In the subsequent qRT-PCR analysis, hBM-MSCs showed a significantly higher expression than did hASCs in following genes: CXCL12, CXCL16, TGF-ß2, SMAD3, COLLAGEN 14A1, and SCLERAXIS (p < 0.05, regardless of BM donor age). In the case of CXCL12, the difference between hASC and hBM-MSC was significant only for younger BM donors, whereas for COLLAGEN 14A1-only for elder BM donors. PPARγ displayed a higher expression in hASCs compared to hBM-MSCs. In regard to CXCL6, MOHAWK, and RUNX2 gene expression, no statistically significant differences between groups were observed. CONCLUSIONS: In the context of cell-based therapy for tendinopathies, bone marrow appears to be a more attractive source of MSCs than does adipose tissue. The age of cell donors seems to be less important than cell source, although cells from elder donors show slightly higher basal tenogenic potential than do cells from younger donors.

Targeting the thioredoxin system as a novel strategy against B-cell acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR-ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP-ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP-ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP-ALL cell lines and pediatric and adult BCP-ALL primary cells, including primary cells cocultured with bone marrow-derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL-rearranged patient-derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP-ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti-BCP-ALL drugs should be continued.

Difficulties in identifying the bacterial species from the genus Clostridium in a case of injury-related osteitis.

Most Clostridium species are part of saprophytic microflora in humans and animals; however, some are well-known human pathogens. We presented the challenges in identifying the Clostridium species isolated from a patient with an infected open dislocation of the proximal interphalangeal joint of the fourth digit of the right hand. The clinical materials were intraoperative samples collected from a patient diagnosed with an injury-related infection, with soft tissue loss and tendon sheath involvement. The available biochemical, molecular, and genetic techniques were used in identifying the isolated bacteria. The isolated bacterium was shown to have low biochemical activity; hence, it was not definitively identified via biochemical tests Api 20A or Rapid 32A. Vitek 2 and mass spectrometry methods were equally inconclusive. Clostridium tetani infection was strongly suspected based on the bacterium's morphology and the appearance of its colonies on solid media. It was only via the 16S rRNA sequencing method, which is non-routine and unavailable in most clinical laboratories, that this pathogen was excluded. Despite appropriate pre-laboratory procedures, which are critical for obtaining reliable test results, the routine methods of anaerobic bacterium identification are not always useful in diagnostics. Diagnostic difficulties occur in the case of environment-derived bacteria of low or not fully understood biological activity, which are absent from databases of automatic bacterial identification systems.

Orthopedic infections caused by obligatory anaerobic Gram-negative rods: report of two cases.

Anaerobic bone and joint infections are uncommon, although the number of anaerobic infections is presumably underestimated because of difficulties with isolation and identification of obligate anaerobes. This study describes two cases of complicated Bacteroides fragilis peri-implant infection of the lumbar spine, infection of the hip and osteomyelitis. Bacteria were identified with the use of a mass spectrometer, VITEK MS system. Drug susceptibility was performed with the use of E-test. The EUCAST breakpoints were used for interpretation with B. fragilis ATCC 25285 as a control. In the two described cases clinical samples were collected for microbiological examination intraoperatively and simultaneously empirical treatment was applied. B. fragilis was isolated in monoculture or in a combination with other bacteria. The treatment was continued according to the susceptibility tests. In a case one clindamycin failure was observed and clindamycin resistance of the isolate was likely due to inadequate time of therapy. Difficulties in collecting an adequate samples and culturing anaerobic bacteria cause that not all infections are properly recognized. In a successful therapy, identification and determination of the susceptibility of the pathogen are essential as well as an appropriate surgical debridement.

Economic Analysis of 4221 Revisions Due to Periprosthetic Joint Infection in Poland.

BACKGROUND: Periprosthetic joint infection (PJI) is one of the most severe complications of total hip (THA) and total knee (TKA) arthroplasty. The aim of the study is to determine the number and type of hip and knee prosthesis revisions in Poland performed due to infection and reimbursement of the cost of septic revisions and to compare the costs of septic and aseptic revisions in Poland and other countries. MATERIAL AND METHODS: The data published for the period 2009-2013 by the National Health Fund (NHF) were analysed and the average cost of septic and aseptic revisions was calculated. RESULTS: In the years 2009-2013, a total of 260,030 hip and knee arthroplasties including 23,027 revisions (incl. 4,221 septic) were performed in Poland. In 2013, septic revisions accounted for 1.38% of all hip replacement procedures, 2.56% of all knee replacement procedures and 14.67% of all hip revisions and 30.23% of all knee revisions. In 2013, the difference between the average cost incurred by the hospital and the NHF refund for septic revision due to PJI was at least €238 and the cost-refund gap for the entire year was €219198. CONCLUSION: 1. The system of reporting periprostheticjoint infections currently in use in Poland does not adequately reflect the current classification of PJI and reimbursement for septic revision of joint prosthesis does not match the actual costs. 2. The Polish DRG system does not distinguish between early and late PJI and fails to acknowledge basic guidelines for infection treatment currently followed in Poland and worldwide. 3. According to the DRG system, patients requiring different treatment are placed in one category. 4. Until the year 2013, the less expensive treatment of early infections had been reimbursed on the same basis as the more costly two-stage revision procedures.

Comparison of Bone Preserving and Radical Surgical Treatment in 32 Cases of Calcaneal Osteomyelitis.

Introduction. Radical procedures like calcanectomy and amputation performed for calcaneal osteomyelitis are regarded as effective in eradication of infection even though potentially functionally disabling. Bone sparing procedures offer better functional result at the expense of potentially worse infection control. The aim of the study has been to assess the influence of the surgical radicalism as much as the extent of bone infection on the final outcome in the surgical therapy of chronic calcaneal osteomyelitis (CO). Material and method. 32 patients with chronic CO have comprised the group under study: 8 with superficial type, 12 localised type and 12 with diffuse type according to Cierny-Mader classification. The aim of the treatment was to heal infection, preserve the heel shape and achieve good skin coverage over the calcaneus. The therapy consisted of 9 debridement surgeries with or without flaps, 8 drilling-operations of the calcaneus with application of collagen-gentamicin-sponge in bore holes, 15 partial and 2 total calcanectomies, and 4 below-the knee amputations. Results. The healing of infection and wound has been achieved after 7 of 9 debridements, 6 of 8 drilling-operations, 13 of 15 partial and all total calcanectomies. Conclusion. Bone preserving operations in chronic calcaneal osteomyelitis provided inferior infection control (76,47% vs 88,24%) and worse patient satisfaction (88,24% vs 100%) and almost camparable ambulation (100% vs 93,33%). Drilling of the calcaneus with application of collagen sponge containing gentamicin performed in chronic diffuse calcaneal osteomyelitis seems to offer a viable alternative to partial or radical calcanectomy. LEVEL OF EVIDENCE: V.

Uremic toxins impair human bone marrow-derived mesenchymal stem cells functionality in vitro.

Mesenchymal stem cells (MSCs) are becoming therapeutic agents of interest in many areas of medicine, including renal diseases and kidney transplantations. However, the effect of uremia on cell properties is still unclear. Therefore, we examined the in vitro influence of uremic toxins, p-cresol (PC) and indoxyl sulfate (IS), on human bone marrow-derived MSC functionality. Cultured MSCs were treated with PC and IS at concentrations corresponding to subsequent stages of chronic kidney disease. Cell viability was characterized by metabolic activity (MTT assay) and proliferation rate (BrdU assay). Apoptosis (Annexin V test) and cell membrane damage (LDH assay) were also tested. MSC secretory properties were determined by measuring cytokine/growth factor levels in media from toxin-treated cells (ELISA). Uremic concentrations of PC and IS resulted in significant inhibition of MSC metabolic activity and proliferation. Toxins did not induce apoptosis, but damaged cell membranes. MSC paracrine activity was also altered - a decrease of VEGF and TGF-ß1 levels and an increase in IGF-1 and IL-8 secretion was detected. Presented data indicate a negative influence of uremic toxins on functional characteristics of human bone marrow-derived MSCs. Therefore, their use as autologous therapeutic agents for kidney disease may be questionable and requires further investigations. The observed phenomenon may be attributable to many other MSC therapies, because of the high prevalence of chronic kidney disease in adult population.

Treatment of solitary bone cysts with allogenic bone graft and platelet-rich plasma. A preliminary report.

The treatment of solitary bone cysts remains controversial. The high recurrence rate after operative treatment calls for the search of new effective treatment methods. The aim of this study was to evaluate the outcomes of treatment of solitary bone cysts with platelet-rich plasma (PRP) and allogenic bone grafts. The study group consisted of 9 patients (4 males and 5 females) with the diagnosis of solitary bone cyst. Their mean age was 12 years and 2 months (range: 6 to 17 years). All patients were symptomatic in routine daily activities. There was a coexisting fracture within the cyst wall in three patients. The operative procedure included removal of the cyst wall soft-tissue lining and filling of the cavity with deep frozen, gamma irradiated morselized allogenic bone grafts mixed with PRP. The PRP was prepared preoperatively with the GPS System (Gravitational Platelet Separation System, Biomet Merck). In six patients with an existing or impending fracture in particularly large cysts, fixation was used. For the cysts located close to the epiphysis (2 femurs, 1 humerus) we used an IM nail or DHS. For three cysts located in the diaphyseal region of the humerus we used an Ilizarov fixator. Three cysts with lower risk of impending fracture were left without fixation. The mean follow-up period was 19.5 months (range: 12 to 30). We noted no procedure-related complications and no refracture during the observation period. Bleeding from the wound was minimal, possibly as a result of PPP use. The wounds healed without swelling or excessive scar formation. After 12 months all the cysts were completely filled with new bone and were staged as Neer stage I. All the patients were asymptomatic. The use of PRP with allogenic bone grafts appears as a promising method for the treatment of solitary bone cysts. Further studies on larger patients series with longer follow-up will be necessary to answer the question whether this method will provide a lower recurrence rate compared to other forms of treatment.

PNF-based rehabilitation in patients with severe haemophilic arthropathy--case study.

Haemophilia is a congenital haemorrhagic diathesis that in its most severe form leads to a seriously disabling arthropathy as a result of recurring intraarticular bleeding. Within the last couple of years there have been significant advances in the treatment of haemophiliacs that are helping to prevent crippling musculoskeletal deformities, but a relatively large number of young adults still present with advanced arthropathic changes when treatment was started too late or has failed to prevent changes from taking place. We present the case of a 44-year-old male with advanced haemophilic arthropathy of the knee and ankle joints, who, during 8 weeks of therapy that consisted of physical therapy and rehabilitation using PNF techniques (Proprioceptive Neuromuscular Facilitation), achieved mobility improvement according to the SPPB scale (Short Physical Performance Battery Test) and a reduction of pain in the knee and ankle joints, according to a VAS scale. The therapeutic regimen proved to be an effective method improving the locomotor function of the patient.