High-Speed Fluorescence Imaging Corroborates Biological Data on the Influence of Different Nozzle Types on Cell Spray Viability and Formation.

Treating severe dermal disruptions often presents significant challenges. Recent advancements have explored biological cell sprays as a promising treatment, but their success hinges on efficient cell delivery and complete wound coverage. This requires a good spray distribution with a small droplet size, high particle number, and ample surface coverage. The type of nozzle used with the spray device can impact these parameters. To evaluate the influence of different nozzles on spray characteristics, we compared air-assisted and unassisted nozzles. The unassisted nozzle displayed small particle size, high particle number, good overall coverage, high cell viability, preserved cell metabolic activity, and low cytotoxicity. Air-assisted nozzles did not perform well regarding cell viability and metabolic activity. Flow visualization analysis comparing two different unassisted nozzles using high-speed imaging (100 kHz frame rate) revealed a tulip-shaped spray pattern, indicating optimal spray distribution. High-speed imaging showed differences between the unassisted nozzles. One unassisted nozzle displayed a bi-modal distribution of the droplet diameter while the other unassisted nozzle displayed a mono-modal distribution. These findings demonstrate the critical role of nozzle selection in successful cell delivery. A high-quality, certified nozzle manufactured for human application omits the need for an air-assisted nozzle and provides a simple system to use with similar or better performance characteristics than those of an air-assisted system.

Peracetic acid sterilized tendon and ligament allografts for knee reconstruction : For anterior cruciate ligament (ACL), posterior cruciate ligament (PCL) and complex knee surgery.

BACKGROUND: The use of allografts and autografts has been met with mixed views on whether allografts are a suitable alternative to autografts. QUESTION: We aimed to investigate if chemically sterilized allografts show similar rerupture rates to those reported in the literature for allografts and autografts in anterior (ACL) and posterior cruciate ligaments (PCL) and complex knee surgery. MATERIALS AND METHODS: Retrospective data on knee reconstructions performed between 2011 and 2015 with tendon/ligamnet allografts sterilized with peracetic acid were collected in the form of a questionnaire. The inclusion criteria of 2 years for each patient were met by 38 patients, representing 22 ACL reconstructions, 5 PCL reconstructions, 3 OTHER surgeries, including the Larson technique and medial patellofemoral ligament (MPFL) reconstruction and 8 COMPLEX surgeries. The main endpoints were rerupture and complication rate. Secondary endpoints included stability of the knee (Lachman test, Pivot shift test) and the range of motion. RESULTS: The rerupture rate was 7.9% (3 grafts). Reruptures only occurred in the ACL group. No reruptures were observed in the PCL, OTHER and COMPLEX surgery groups. Stability improved significantly after surgery and the range of motion returned to values similar to that of healthy knees. CONCLUSIONS: Tendon allografts sterilized with peracetic acid show promising low rerupture rates and good clinical scores and the results are comparable to the literature on autografts and other allografts.

Allografts for partial meniscus repair: an in vitro and ex vivo meniscus culture study.

The purpose of this study was to evaluate the treatment potential of a human-derived demineralized scaffold, Spongioflex® (SPX), in partial meniscal lesions by employing in vitro models. In the first step, the differentiation potential of human meniscal cells (MCs) was investigated. In the next step, the ability of SPX to accommodate and support the adherence and/or growth of MCs while maintaining their fibroblastic/chondrocytic properties was studied. Control scaffolds, including bovine collagen meniscus implant (CMI) and human meniscus allograft (M-Allo), were used for comparison purposes. In addition, the migration tendency of MCs from fresh donor meniscal tissue into SPX was investigated in an ex vivo model. The results showed that MCs cultured in osteogenic medium did not differentiate into osteogenic cells or form significant calcium phosphate deposits, although AP activity was relatively increased in these cells. Culturing cells on the scaffolds revealed increased viability on SPX compared to the other scaffold materials. Collagen I synthesis, assessed by ELISA, was similar in cells cultured in 2D and on SPX. MCs on micro-porous SPX (weight >0.5 g/cm3) exhibited increased osteogenic differentiation indicated by upregulated expression of ALP and RUNX2, while also showing upregulated expression of the chondrogen-specific SOX9 and ACAN genes. Ingrowth of cells on SPX was observed after 28 days of cultivation. Overall, the results suggest that SPX could be a promising biocompatible scaffold for meniscal regeneration.

Anatomical acromioclavicular joint stabilization with chemically sterilized tendon allografts: A retrospective study.

Background: The purpose of this retrospective study was to analyze whether chemically sterilized tendon allografts perform as well as other non-sterilized allografts and autografts as described in the literature for anatomical acromioclavicular joint stabilization for the treatment of Rockwood III-V. Allografts are still described as a factor for higher re-rupture rates. Methods: Retrospective data were collected from 21 acromioclavicular joint stabilizations performed by a single surgeon and performed between 2011 and 2014 using sterilized semitendinosus allografts. The primary endpoints were re-rupture and complication rates. Secondary endpoints were AC-joint stability, pain level, return to work and sport and the range of motion. Results: No re-ruptures occurred during the mean follow-up time of 33 months. Zero complications occurred directly after surgery, but three complications later than three weeks after surgery. All cases resolved without further surgery. After surgery, stability significantly improved for all patients. Post-surgery, 19 patients had stable acromioclavicular joints and only two patients showed minor instabilities. Range of motion returned to the range of the healthy shoulders for all patients. Conclusion: Chemically sterilized semitendinosus allograft use for anatomic AC-joint stabilization is equivalent to the use of other allografts or autografts and required no hardware removal. No donor age or graft size dependence was observed, due to zero re-ruptures.

Analysis of the Ability of Different Allografts to Act as Carrier Grafts for Local Drug Delivery.

Bone defects and infections pose significant challenges for treatment, requiring a comprehensive approach for prevention and treatment. Thus, this study sought to evaluate the efficacy of various bone allografts in the absorption and release of antibiotics. A specially designed high-absorbency, high-surface-area carrier graft composed of human demineralized cortical fibers and granulated cancellous bone (fibrous graft) was compared to different human bone allograft types. The groups tested here were three fibrous grafts with rehydration rates of 2.7, 4, and 8 mL/g (F(2.7), F(4), and F(8)); demineralized bone matrix (DBM); cortical granules; mineralized cancellous bone; and demineralized cancellous bone. The absorption capacity of the bone grafts was assessed after rehydration, the duration of absorption varied from 5 to 30 min, and the elution kinetics of gentamicin were determined over 21 days. Furthermore, antimicrobial activity was assessed using a zone of inhibition (ZOI) test with S. aureus. The fibrous grafts exhibited the greatest tissue matrix absorption capacity, while the mineralized cancellous bone revealed the lowest matrix-bound absorption capacity. For F(2.7) and F(4), a greater elution of gentamicin was observed from 4 h and continuously over the first 3 days when compared to the other grafts. Release kinetics were only marginally affected by the varied incubation times. The enhanced absorption capacity of the fibrous grafts resulted in a prolonged antibiotic release and activity. Therefore, fibrous grafts can serve as suitable carrier grafts, as they are able to retain fluids such as antibiotics at their intended destinations, are easy to handle, and allow for a prolonged antibiotic release. Application of these fibrous grafts can enable surgeons to provide longer courses of antibiotic administration for septic orthopedic indications, thus minimizing infections.

Allografts: expanding the surgeon's armamentarium.

In Germany, bone allografts are widely used and their application in clinics has increased over the years. Successful use of allografts depends on many factors such as the procurement, processing, sterilization and the surgeon's surgical experience. Tissue banks have provided safe and sterile allografts for decades ranging from hard to soft tissue. Allografts are obtained from various tissues such as bone, tendon, amniotic membrane, meniscus and skin. An advantage of allografts is their wide applicability that has never been limited by indication restrictions thus providing a huge benefit for surgeon's. The use of the correct allograft in different indications is extremely important. Thereby surgeons have access to various allograft forms such as mineralized, demineralized, freeze-dried, paste, powder, chips strips and putty. The vast options of allografts allow surgeon's to use allografts in indications they deem fit. Currently, the application of allografts is at the discretion of the expert surgeon. However, regulations are often changed locally or internationally and may impact/limit allograft use to certain indications. Here, we report the different indications where our peracetic acid (PAA) sterilised bone allografts were used as well as general literature on bone allograft use in other indications.

Influence of peracetic acid-ethanol sterilisation on the biomechanical properties of human meniscus transplants.

PURPOSE: Meniscus allograft transplantation (MAT) is a possible treatment for patients suffering with pain after meniscectomy. Here, peracetic acid (PAA) sterilised meniscus transplants were investigated on whether they would provide an adequate alternative to fresh-frozen transplants in their viscoelastic and mechanical properties. METHODS: In this analysis, 31 menisci donors (26 male and 5 female) were included. The average donor age was 49.87 years, ranging from 32 to 65 years. Menisci of matched pairs of knees underwent chemical sterilisation while counterparts were left fresh-frozen. Stiffness and load to failure were determined via suture retention. Further menisci were analysed while attached to the tibial bone block using a novel test device to mimic physiological load distribution. Meniscus relaxation, stiffness and failure loads were determined. Histology and biphasic properties of the menisci were examined and results were analysed using paired t-tests. RESULTS: A novel custom built test device allowed the application of physiological loads for suture retention testing and revealed no significant differences between PAA sterilised (14.85 ± 4.46 N/mm, 50.49 ± 17.01 N) and fresh-frozen (18.26 ± 4.46 N/mm, 59.49 ± 21.07 N) regarding stiffness and failure load, respectively. Furthermore, initial 200 N loading showed significantly higher strain in sterilised menisci (18.87 ± 1.56) compared to fresh frozen (13.81 ± 1.04). Load relaxation experiments demonstrated significantly lower relaxation for sterilised menisci (77.71 ± 1.62) compared to fresh-frozen (89.11 ± 1.00, p-value < 0.0001). CONCLUSION: Peracetic acid sterilised human menisci performed equally to fresh-frozen counterparts in a suture retention test and in physiological failure testing providing an adequate alternative. However, meniscus relaxation, biphasic properties and strain were shown to be significantly different between the groups. A common problem of MAT is graft extrusion or shrinkage, therefore the parameters measured here should be considered and may influence meniscus extrusion after transplantation. LEVEL OF EVIDENCE: n/a (experimental study).

Neuronal impairment following chronic Toxoplasma gondii infection is aggravated by intestinal nematode challenge in an IFN-γ-dependent manner.

BACKGROUND: It has become increasingly evident that the immune and nervous systems are closely intertwined, relying on one another during regular homeostatic conditions. Prolonged states of imbalance between neural and immune homeostasis, such as chronic neuroinflammation, are associated with a higher risk for neural damage. Toxoplasma gondii is a highly successful neurotropic parasite causing persistent subclinical neuroinflammation, which is associated with psychiatric and neurodegenerative disorders. Little is known, however, by what means neuroinflammation and the associated neural impairment can be modulated by peripheral inflammatory processes. METHODS: Expression of immune and synapse-associated genes was assessed via quantitative real-time PCR to investigate how T. gondii infection-induced chronic neuroinflammation and associated neuronal alterations can be reshaped by a subsequent acute intestinal nematode co-infection. Immune cell subsets were characterized via flow cytometry in the brain of infected mice. Sulfadiazine and interferon-γ-neutralizing antibody were applied to subdue neuroinflammation. RESULTS: Neuroinflammation induced by T. gondii infection of mice was associated with increased microglia activation, recruitment of immune cells into the brain exhibiting Th1 effector functions, and enhanced production of Th1 and pro-inflammatory molecules (IFN-γ, iNOS, IL-12, TNF, IL-6, and IL-1ß) following co-infection with Heligmosomoides polygyrus. The accelerated cerebral Th1 immune response resulted in enhanced T. gondii removal but exacerbated the inflammation-related decrease of synapse-associated gene expression. Synaptic proteins EAAT2 and GABAAα1, which are involved in the excitation/inhibition balance in the CNS, were affected in particular. These synaptic alterations were partially recovered by reducing neuroinflammation indirectly via antiparasitic treatment and especially by application of IFN-γ-neutralizing antibody. Impaired iNOS expression following IFN-γ neutralization directly affected EAAT2 and GABAAα1 signaling, thus contributing to the microglial regulation of neurons. Besides, reduced CD36, TREM2, and C1qa gene expression points toward inflammation induced synaptic pruning as a fundamental mechanism. CONCLUSION: Our results suggest that neuroimmune responses following chronic T. gondii infection can be modulated by acute enteric nematode co-infection. While consecutive co-infection promotes parasite elimination in the CNS, it also adversely affects gene expression of synaptic proteins, via an IFN-γ-dependent manner.

A Novel Non-invasive Method to Detect RELM Beta Transcript in Gut Barrier Related Changes During a Gastrointestinal Nematode Infection.

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-ß, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-ß was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-ß expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection.

Toxoplasma Co-infection Prevents Th2 Differentiation and Leads to a Helminth-Specific Th1 Response.

Nematode infections, in particular gastrointestinal nematodes, are widespread and co-infections with other parasites and pathogens are frequently encountered in humans and animals. To decipher the immunological effects of a widespread protozoan infection on the anti-helminth immune response we studied a co-infection with the enteric nematode Heligmosomoides polygyrus in mice previously infected with Toxoplasma gondii. Protective immune responses against nematodes are dependent on parasite-specific Th2 responses associated with IL-4, IL-5, IL-13, IgE, and IgG1 antibodies. In contrast, Toxoplasma gondii infection elicits a strong and protective Th1 immune response characterized by IFN-γ, IL-12, and IgG2a antibodies. Co-infected animals displayed significantly higher worm fecundity although worm burden remained unchanged. In line with this, the Th2 response to H. polygyrus in co-infected animals showed a profound reduction of IL-4, IL-5, IL-13, and GATA-3 expressing T cells. Co-infection also resulted in the lack of eosinophilia and reduced expression of the Th2 effector molecule RELM-ß in intestinal tissue. In contrast, the Th1 response to the protozoan parasite was not diminished and parasitemia of T. gondii was unaffected by concurrent helminth infection. Importantly, H. polygyrus specific restimulation of splenocytes revealed H. polygyrus-reactive CD4+ T cells that produce a significant amount of IFN-γ in co-infected animals. This was not observed in animals infected with the nematode alone. Increased levels of H. polygyrus-specific IgG2a antibodies in co-infected mice mirrored this finding. This study suggests that polarization rather than priming of naive CD4+ T cells is disturbed in mice previously infected with T. gondii. In conclusion, a previous T. gondii infection limits a helminth-specific Th2 immune response while promoting a shift toward a Th1-type immune response.