Musculoskeletal impairments, especially cartilage and meniscus lesions, are some of the major contributors to disabilities. Thus, novel tissue engineering strategies are being developed to overcome these issues. In this study, the aim was to investigate the biocompatibility, in vitro and in vivo, of a thermosensitive, injectable chitosan-based hydrogel loaded with three different primary mesenchymal stromal cells. The cell types were human adipose-derived mesenchymal stromal cells (hASCs), human bone marrow stem cells (hBMSCs), and neonatal porcine infrapatellar fat-derived cells (IFPCs). For the in vitro study, the cells were encapsulated in sol-phase hydrogel, and then, analyzed via live/dead assay at 1, 4, 7, and 14 days to compare their capacity to survive in the hydrogel. To assess biocompatibility in vivo, cellularized scaffolds were subcutaneously implanted in the dorsal pouches of nude mice and analyzed at 4 and 12 weeks. Our data showed that all the different cell types survived (the live cell percentages were between 60 and 80 at all time points in vitro) and proliferated in the hydrogel (from very few at 4 weeks to up to 30% at 12 weeks in vivo); moreover, the cell-laden hydrogels did not trigger an immune response in vivo. Hence, our hydrogel formulation showed a favorable profile in terms of safety and biocompatibility, and it may be applied in tissue engineering strategies for cartilage and meniscus repair.
BACKGROUND Humoral response after two doses of mRNA-based SARS-CoV-2 vaccines is weak in kidney transplant recipients (KTRs) [1]. Of concern, even a third dose of the mRNA-1273 vaccine induced a suboptimal humoral response in a cohort of KTRs who did not respond after two doses [2]. However, the assessment also of the T-cell immune response of mRNA vaccines in KTRs is limited in the literature [3]. Herein, we report the evaluation of humoral response induced after a third dose of the mRNA-1273 vaccine in a cohort of KTRs and T-cell immune responses in not responders to the third dose. METHODS Observational cohort data were collected from KTRs actively followed up in our outpatient clinic, with no history of COVID-19 infection, who received a third mRNA-1273 vaccine dose, 6 months after the second dose, as per suggested local policy. The primary endpoint was the humoral response provided at least 4 weeks after the third dose compared with the humoral response after the second dose. Anti-S1-RBD IgG antibodies were determined using a fluoroimmunoenzymatic method (Thermo Fisher Scientific), and the quantitative result expressed in BAU/mL (reference interval: <28 Negative; 28–40 Borderline; >40 Positive; linear range between 0.7 and >1632 according to the manufacturer). In seronegative and borderline KTRs after the third dose, an INFγ-release assay (IGRA) [Euroimmun, Lubeck, Germany] was used to detect T-cell immune responses. A patient result was considered negative, borderline and positive when IFNγ values were respectively <100, 100–200 and >200 mIU/mL. RESULTS Sixty KTRs received a third dose of the mRNA-1273 vaccine. Overall, we obtained the antibody titre in 57 KTRs at a median of 23 days (IQR: 22–31) after the second dose and 23 days (IQR: 21–26) after the third dose. After the second dose, positive antibody titres were detectable in 28 KTRs (49%), and 2 KTRs (4%) had a borderline positivity. While after third dose, positive and borderline antibody responses were observed in 40 (70%) and 4 (7%) KTRs, respectively. Among all 57 KTRs, the median anti-S1-RBD IgG titre significantly increased after the third dose ( 448 versus 39 BAU/mL; P = 0.0018; Mann–Whitney test). While in 28 KTRs already seropositive after the second dose, the median antibody titre increased from 556 to >1632 BAU/mL (P = 0.0285; Mann–Whitney test). Figure 1 shows the kinetics of anti-S1-RBD IgG titres after the second and the third dose for all the 57 KTRs. Among 17 KTRs with negative and borderline humoral responses after the third dose, IFNγ values were positive and borderline in only 1 (6%) and 1 (6%) KTRs, respectively. The median IFNγ value was 22 mIU/mL (IQR: 0–1014). The 34 KTRs receiving mycophenolate mofetil (MMF) were less likely to develop adequate immune responses than the 23 KTRs not receiving MMF; 12 KTRs (35%) receiving MMF had no antibody and IFNγ positive response after the third dose in comparison to 4 KTRs (17%) not receiving MMF, and the median anti-S1-RBD IgG titre beyond the two groups after the third dose was statistically different (205 versus > 1632; P = 0.0046; Mann–Whitney test). CONCLUSIONS In this study, the third dose of the mRNA-1273 vaccine increases the rate of positive antibody responses in non-responders KTRs after the second dose, and improves the magnitude of these responses in already seropositive KTRs. However, a fraction of KTRs still lacks protective antibody titres and T-cell responses after a third dose, with mycophenolate mofetil to be associated with poor immune responses. Alternative vaccination protocols are needed to protect this high-risk group.
BACKGROUND Advance care planning (ACP) enables competent patients to define goals and preferences for future treatments and care, to discuss these goals and preferences with relatives, and if appropriate to record these preferences [1]. After many years of political and social debates, in December 2017 the first advance directive and care planning legislation was approved in Italy. In the last years, several national recommendations released by political institutions and medical associations have underlined the importance of conducting ACP with chronic patients, included dialyses patients. Nevertheless, citizens’ awareness of these issues is scarce as well as the integration of the ACP process into clinical routine [2]. METHOD Since November 2020, our nephrology unit adopted a protocol aimed to conduct ACP interventions with dialysis patients. Firstly, all patients were informed about the possibility to taking part in one or more ACP conversations with their nephrologist. Secondly, a semi-structured guide to the conversation was created in order to support physicians in conducting the ACP intervention. The guide is divided into three sections: (1) exploring patient's understanding of the disease and his/her hopes and life goals; (2) patient's social relationships; (3) preferences for future treatments and care (e.g. CPR, dialysis, palliative care, etc). Finally, patients along with nephrologist and their relatives can document their preferences. RESULTS Of 110 haemodialysis patients, 40 treated in our centre asked for an ACP intervention and filled-in an advance directive. Specifically, no patients required an immediate discontinuation of dialysis; nevertheless, 70% of them stated that would not like to continue with dialysis in case he/she was no longer able to self-determine (e.g. permanent loss of capacity to communicate with others). Most of patients (90%) have appointed a personal representative (usually their partner). In the eventuality of cardiac arrest, 55% of patients asked for cardiopulmonary resuscitation. Pain control and palliative care was the overall most cited topic. The fear of being a burden on family members has been frequently expressed by not independent patients. Regarding the last phase of life, 75% of patients expressed the desire to stay at home, the others in hospice. CONCLUSION ACP process is very recent in Italy and not well known by patients and healthcare providers (HCPs). As a result, discussions about future treatments and care are rarely included in the clinical routine with haemodialysis patients. Training of HCPs on these issues as well as the use of procedures tailored to the Italian context for conducting ACP interventions is needed.
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