Prevention of Transition from Acute Kidney Injury to Chronic Kidney Disease Using Clinical-Grade Perinatal Stem Cells in Non-Clinical Study.

Acute kidney injury (AKI) is widely recognized as a precursor to the onset or rapid progression of chronic kidney disease (CKD). However, there is currently no effective treatment available for AKI, underscoring the urgent need for the development of new strategies to improve kidney function. Human placental mesenchymal stromal cells (hpMSCs) were isolated from donor placentas, cultured, and characterized with regard to yield, viability, flow cytometry, and potency. To mimic AKI and its progression to CKD in a rat model, a dedicated sensitive non-clinical bilateral kidney ischemia-reperfusion injury (IRI) model was utilized. The experimental group received 3 × 105 hpMSCs into each kidney, while the control group received IRI and saline and the untreated group received IRI only. Urine, serum, and kidney tissue samples were collected over a period of 28 days. The hpMSCs exhibited consistent yields, viability, and expression of mesenchymal lineage markers, and were also shown to suppress T cell proliferation in a dose-dependent manner. To ensure optimal donor selection, manufacturing optimization, and rigorous quality control, the rigorous Good Manufacturing Practice (GMP) conditions were utilized. The results indicated that hpMSCs increased rat survival rates and improved kidney function by decreasing serum creatinine, urea, potassium, and fractionated potassium levels. Furthermore, the study demonstrated that hpMSCs can prevent the initial stages of kidney structural fibrosis and improve kidney function in the early stages by mitigating late interstitial fibrosis and tubular atrophy. Additionally, a robust manufacturing process with consistent technical parameters was established.

Characterization of tissue engineered cartilage products: Recent developments in advanced therapy.

Legislative requirements for the quality of pharmacological agents underwent certain evolution when new type of therapies emerged. This relates to cell based medicines, such as tissue engineered cartilage products (TECP) which are increasingly developed as new modalities for widely prevalent orthopaedic disorders. Although quality measures for TECP are subject to the same general regulatory quality requirements, combination of cellular and scaffold substances requires definition of specific characteristics in vitro that are highly relevant to potency and efficacy of the newly designed medicinal product. One of the specific issues in designing cell based medicines is the fact that the biological activity of active substance, or cells, usually is altered after seeding them on a three-dimensional scaffold. Newly acquired features of the TECP are influenced by chemical, physical and mechanical characteristics of the scaffolds. A vast array of analytical methods has been employed to measure efficacy and potency of TECP in cartilage regeneration studies in vitro. Designing specific physical characteristics of scaffolds may become essential part influencing pharmacological activity of cell based medicinal products, and discern TECP from typical pharmacological products. As an example, increasingly growing popularity of three-dimensional printing that utilizes direct laser writing technique provides an opportunity to improve efficacy of the final TECP. This review is intended to provide brief summary of current approaches used to characterize cells and scaffolds in vitro before and after combination into TECP. Validating TECP as pharmacological agents with unique biological and physical characteristics may broaden their clinical application.

Impact of storage conditions on electromechanical, histological and histochemical properties of osteochondral allografts.

BACKGROUND: Osteochondral allograft transplantation has a good clinical outcome, however, there is still debate on optimization of allograft storage protocol. Storage temperature and nutrient medium composition are the most critical factors for sustained biological activity of grafts before implantation. In this study, we performed a time-dependent in vitro experiment to investigate the effect of various storage conditions on electromechanical, histological and histochemical properties of articular cartilage. METHODS: Osteochondral grafts derived from goat femoral condyles were frozen at -70 °C or stored at 4 °C and 37 °C in the medium supplemented with or without insulin-like growth factor-1 (IGF-1). After 14 and 28 days the cartilage samples were quantitatively analysed for electromechanical properties, glycosaminoglycan distribution, histological structure, chondrocyte viability and apoptosis. The results were compared between the experimental groups and correlations among different evaluation methods were determined. RESULTS: Storage at -70 °C and 37 °C significantly deteriorated cartilage electromechanical, histological and histochemical properties. Storage at 4 °C maintained the electromechanical quantitative parameter (QP) and glycosaminoglycan expression near the normal levels for 14 days. Although hypothermic storage revealed reduced chondrocyte viability and increased apoptosis, these parameters were superior compared with the storage at -70 °C and 37 °C. IGF-1 supplementation improved the electromechanical QP, chondrocyte viability and histological properties at 37 °C, but the effect lasted only 14 days. Electromechanical properties correlated with the histological grading score (r = 0.673, p < 0.001), chondrocyte viability (r = -0.654, p < 0.001) and apoptosis (r = 0.416, p < 0.02). In addition, apoptosis correlated with glycosaminoglycan distribution (r = -0.644, p < 0.001) and the histological grading score (r = 0.493, p = 0.006). CONCLUSIONS: Our results indicate that quality of allografts is better preserved at currently established 4 °C storage temperature. Storage at -70 °C or at 37 °C is unable to maintain cartilage function and metabolic activity. IGF-1 supplementation at 37 °C can enhance chondrocyte viability and improve electromechanical and histological properties of the cartilage, but the impact persists only 14 days. The correlations between cartilage electromechanical quantitative parameter (QP) and metabolic activity were detected. Our findings indicate that non-destructive assessment of cartilage by Arthro-BST is a simple and reliable method to evaluate allograft quality, and could be routinely used before implantation.

Arthroscopic Implantation of a Cell-Free Bilayer Scaffold for the Treatment of Knee Chondral Lesions: A 2-Year Prospective Study.

OBJECTIVE: The main objective of this study is to assess the safety and clinical efficacy of a cell-free bilayer scaffold (MaioRegen Chondro+ by Fin-Ceramica) in patients affected by chondral knee lesions of different origin and localization. DESIGN: Thirty-one patients with focal chondral lesions of the knee were arthroscopically treated with MaioRegen Chondro+. All patients were prospectively evaluated for a minimum of 2 years using the International Knee Documentation Committee (IKDC) Questionnaire and the Tegner Activity Scale. Cartilage repair was assessed based on the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) 2.0 score at 12 months. Follow-up at 36 months was available for 25 out of 31 patients. RESULTS: From baseline to 6-, 12-, and 24-month follow-up, IKDC score significantly improved by 19.5 ± 7.27 (95% confidence interval [CI]: 16.9-22.2, P < 0.001), 30.8 ± 7.63 (95% CI: 28.0-33.6, P < 0.001), and 36.2 ± 8.00 points (95% CI: 33.3-39.2, P < 0.001), respectively. Tegner scores documented a substantial clinical improvement as early as 12 months after surgery (change of -0.6 ± 0.62; 95% CI: -0.8 to -0.4, P < 0.001), reaching the preinjury values. There was a statistically significant increase in the MOCART scores (P < 0.001). Comparable results were observed regardless of preintervention demographic characteristics, lesion site or etiology, or the number of treated sites. Notably, the significant clinical benefit was maintained in a subset of patients who reached 3-year follow-up. No adverse events were reported in the entire analyzed population. CONCLUSION: MaioRegen Chondro+ is a safe and effective device for the treatment of knee chondral lesions, enabling a significant clinical improvement for at least 2 years.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 µg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1ß, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.

Arthroscopic Electromechanical Assessment of Human Articular Cartilage Injury Correlates with ICRS Scores.

PURPOSE: This study aimed to conduct arthroscopic evaluation of cartilage electromechanical properties and establish their correlation with International Cartilage Repair Society (ICRS) grading scores. METHODS: In 18 patients, quantitative parameter (QP) measurements were taken on the weight-bearing surface of the medial femoral condyle. Adjacently, the same site was graded using ICRS scores (0-4). Electromechanical QPs for ICRS grades 0 to 3 were obtained during arthroscopy, while complete grade 4 injuries were assessed using femur cartilage-bone blocks from knee arthroplasty. The QP values for ICRS grades 0 to 2 were compared with grades 3 and 4 using Welch t test. The corresponding QP values were assigned to ICRS grades 0 to 4 and compared using Welch ANOVA (analysis of variance). Pearson's coefficient evaluated QP-ICRS grade relationship. RESULTS: Healthy grade 0 cartilage displayed a mean QP value of 10.5 (±2.8 SD, n = 4). The ICRS grade 1 and grade 2 injuries were associated with QP values of 12 (±0.7, n = 2) and 13.25 (±1.77, n = 2), respectively. The grade 3 defects had QP values of 20.43 (±4.84, n = 4), whereas complete grade 4 defects showed electromechanical values of 30.17 (±2.19, n = 6). Significant differences in QP values were observed between ICRS grades 0 to 2 (mean QP 11.56 ± 2.3, n = 8) and grades 3 and 4 (26.27 ± 6, n = 10; P < 0.0001). Pearson's correlation coefficient of 0.9 indicated a strong association between higher ICRS cartilage injury grades and elevated QP values (P < 0.0001). CONCLUSION: Arthroscopic electromechanical QP assessment robustly correlates with ICRS scores. The QP values for ICRS grades 0 to 2 are significantly lower, compared with grades 3 and 4.

Human Placental Mesenchymal Stem Cells and Derived Extracellular Vesicles Ameliorate Lung Injury in Acute Respiratory Distress Syndrome Murine Model.

This study investigates the therapeutic potential of human placental mesenchymal stem cells (P-MSCs) and their extracellular vesicles (EVs) in a murine model of acute respiratory distress syndrome (ARDS), a condition with growing relevance due to its association with severe COVID-19. We induced ARDS-like lung injury in mice using intranasal LPS instillation and evaluated histological changes, neutrophil accumulation via immunohistochemistry, bronchoalveolar lavage fluid cell count, total protein, and cytokine concentration, as well as lung gene expression changes at three time points: 24, 72, and 168 h. We found that both P-MSCs and EV treatments reduced the histological evidence of lung injury, decreased neutrophil infiltration, and improved alveolar barrier integrity. Analyses of cytokines and gene expression revealed that both treatments accelerated inflammation resolution in lung tissue. Biodistribution studies indicated negligible cell engraftment, suggesting that intraperitoneal P-MSC therapy functions mostly through soluble factors. Overall, both P-MSC and EV therapy ameliorated LPS-induced lung injury. Notably, at the tested dose, EV therapy was more effective than P-MSCs in reducing most aspects of lung injury.

Skeletal muscle-derived stem cells: implications for cell-mediated therapies.

Current advances in stem cell research and innovative biological approaches in the field of tissue engineering and regenerative medicine could eventually translate into prospective clinical applications. Various adult organs and tissues harbor stem and progenitor cells that could potentially be used to repair, regenerate, and restore a variety of different tissues following acute injury or tissue destructive diseases. Skeletal muscle is a very convenient and plentiful source of somatic stem cells. It contains several distinct populations of myogenic stem cells including satellite cells that are mainly responsible for muscle growth and regeneration, and multipotent muscle-derived stem cells (MDSCs). Although both cell populations share some phenotypic similarities, MDSCs display a much greater differentiation potential in vitro and are capable of regenerating various tissues in vivo. Furthermore, these cells not only participate in the regeneration process by differentiating into tissue-specific cell types, but also promote endogenous tissue repair by secreting a multitude of trophic factors. In this article, we describe the biological aspects of MDSC isolation and characterization and provide an overview of potential therapeutic application of these cells for the treatment of cardiac and skeletal muscle injuries and diseases, urological dysfunction, and bone and cartilage defects. We also discuss major challenges and limitations currently faced by MDSC-based therapies that await resolution before these techniques can be applied clinically.

Osteochondral Repair and Electromechanical Evaluation of Custom 3D Scaffold Microstructured by Direct Laser Writing Lithography.

OBJECTIVE: The objective of this study was to assess a novel 3D microstructured scaffold seeded with allogeneic chondrocytes (cells) in a rabbit osteochondral defect model. DESIGN: Direct laser writing lithography in pre-polymers was employed to fabricate custom silicon-zirconium containing hybrid organic-inorganic (HOI) polymer SZ2080 scaffolds of a predefined morphology. Hexagon-pored HOI scaffolds were seeded with chondrocytes (cells), and tissue-engineered cartilage biocompatibility, potency, efficacy, and shelf-life in vitro was assessed by morphological, ELISA (enzyme-linked immunosorbent assay) and PCR (polymerase chain reaction) analysis. Osteochondral defect was created in the weight-bearing area of medial femoral condyle for in vivo study. Polymerized fibrin was added to every defect of 5 experimental groups. Cartilage repair was analyzed after 6 months using macroscopical (Oswestry Arthroscopy Score [OAS]), histological, and electromechanical quantitative potential (QP) scores. Collagen scaffold (CS) was used as a positive comparator for in vitro and in vivo studies. RESULTS: Type II collagen gene upregulation and protein secretion was maintained up to 8 days in seeded HOI. In vivo analysis revealed improvement in all scaffold treatment groups. For the first time, electromechanical properties of a cellular-based scaffold were analyzed in a preclinical study. Cell addition did not enhance OAS but improved histological and QP scores in HOI groups. CONCLUSIONS: HOI material is biocompatible for up to 8 days in vitro and is supportive of cartilage formation at 6 months in vivo. Electromechanical measurement offers a reliable quality assessment of repaired cartilage.

Nondestructive Assessment of Articular Cartilage Electromechanical Properties after Osteochondral Autologous and Allogeneic Transplantation in a Goat Model.

OBJECTIVE: To determine the applicability of a minimally invasive diagnostic device to evaluate the quality of articular cartilage following autologous (OAT) and allogeneic (OCA) osteochondral graft transplantation in goat model. DESIGN: OAT grafts were harvested from lateral femoral condyles (LFCs) and transplanted into osteochondral defects created in medial femoral condyles (MFCs) of contralateral knees. OCA grafts were transplanted into MFC condyles after in vitro storage. Autologous platelet-rich plasma (PRP) was administered intraarticularly after the surgery and at 1 and 2 months postoperatively. OAT and OCA grafts were evaluated macroscopically (Oswestry arthroscopy score [OAS]), electromechanically (quantitative parameter, QP), and histologically (O'Driscoll score, safranin O staining intensity) at 3 and 6 months after transplantation. Results were compared with preoperative graft evaluation. RESULTS: Transplanted cartilage deteriorated within 6 months in all groups. Cartilage quality was better retained in OAT group compared with a decline in OCA group. QP and OAS scores were comparable in OAT and OCA groups at 3 months, but superior in OAT group at 6 months, according to all the methods applied. PRP injections significantly improved QP and OAS score at 6 months compared with 3 months in OAT group. QP moderately correlated with OAS, O'Driscoll score, and safranin O staining intensity. CONCLUSIONS: Grafts did not retain preoperative quality parameters at 6 months follow-up; however, OAT were superior to OCA grafts. PRP may have a beneficial effect on macroscopic and electromechanical properties of cartilage; however, histological improvement is yet to be proved. Electromechanical diagnostic device enables reliable assessment of transplanted cartilage.

Clinical outcome after treatment of single and multiple cartilage defects by autologous matrix-induced chondrogenesis.

PURPOSE: Characterized cartilage lesions have a distinct impact on postoperative clinical outcome, which is still being evaluated. The purpose of this study was to assess the postoperative clinical outcome of autologous matrix-induced chondrogenesis (AMIC) for characterized cartilage lesions. METHODS: Fifteen patients with articular cartilage (AC) defects of the knee were included in the study. AC defects were characterized intraoperatively by International Cartilage Repair Society score. Grade III-IV AC lesions were treated with AMIC; grade I-II lesions were left untreated. Patients were divided into subgroups and clinically evaluated by subjective autologous matrix-induced chondrogenesis (IKDC) and Tegner scores at median follow-up of 4.5 years. RESULTS: Twenty-eight AC defects were diagnosed (1.9/patient). Multiple subgroup had larger diagnosed (7 ± 2.3 cm2, p = 0.022) and untreated (3.1 ± 2.3 cm2, p = 0.012) lesion areas than the single subgroup. Partly treated subgroup had larger untreated defect areas (3.6±2.3 cm2, p = 0.025) than the Treated subgroup. Average subjective IKDC values of total group and individual subgroups improved significantly at follow-up. More patients restored their previous activity levels ( p = 0.026) and had higher incremental subjective IKDC scores ( p = 0.014) in the single subgroup than the multiple subgroup. Diagnosed defect size negatively correlated to subjective IKDC incremental ( r = -0.624, p = 0.023) and postoperative scores ( r = -0.545, p = 0.054) in total group. CONCLUSIONS: AMIC can have a clinically relevant outcome for patients with single or multiple knee AC lesions; however, clinical outcome is superior in patients with a single defect per knee. Patients with single defects returned to previous physical activity levels significantly faster than patients with multiple defects. Diagnosed AC defect areas negatively correlate to clinical improvement at follow-up.

Quantitative Arthroscopic Assessment of Articular Cartilage Quality by Means of Cartilage Electromechanical Properties.

Arthroscopic surgery has grown rapidly in recent decades. Despite accurately diagnosed clinical cases, the previous pain is retained in some patients after the operation, even though no visible chondral lesions are found during the procedure. A minimally invasive arthroscopic method of measuring articular cartilage electromechanical properties enables rapid and reliable intraoperative articular cartilage quality evaluation.

Skeletal Muscle-Derived Stem/Progenitor Cells: A Potential Strategy for the Treatment of Acute Kidney Injury.

Skeletal muscle-derived stem/progenitor cells (MDSPCs) have been thoroughly investigated and already used in preclinical studies. However, therapeutic potential of MDSPCs isolated using preplate isolation technique for acute kidney injury (AKI) has not been evaluated. We aimed to characterize rat MDSPCs, compare them with bone marrow mesenchymal stem cells (BM-MSCs), and evaluate the feasibility of MDSPCs therapy for gentamicin-induced AKI in rats. We have isolated and characterized rat MDSPCs and BM-MSCs. Characteristics of rat BM-MSCs and MDSPCs were assessed by population doubling time, flow cytometry, immunofluorescence staining, RT-PCR, and multipotent differentiation capacity. Gentamicin-induced AKI model in rat was used to examine MDSPCs therapeutic effect. Physiological and histological kidney parameters were determined. MDSPCs exhibited similar immunophenotype, stem cell gene expression, and multilineage differentiation capacities as BM-MSCs, but they demonstrated higher proliferation rate. Single intravenous MDSPCs injection accelerated functional and morphological kidney recovery, as reflected by significantly lower serum creatinine levels, renal injury score, higher urinary creatinine, and GFR levels. PKH-26-labeled MDSPCs were identified within renal cortex 1 and 2 weeks after cell administration, indicating MDSPCs capacity to migrate and populate renal tissue. In conclusion, MDSPCs are capable of mediating functional and histological kidney recovery and can be considered as potential strategy for AKI treatment.

Preclinical study of SZ2080 material 3D microstructured scaffolds for cartilage tissue engineering made by femtosecond direct laser writing lithography.

Over the last decade DLW employing ultrafast pulsed lasers has become a well-established technique for the creation of custom-made free-form three-dimensional (3D) microscaffolds out of a variety of materials ranging from proteins to biocompatible glasses. Its potential applications for manufacturing a patient's specific scaffold seem unlimited in terms of spatial resolution and geometry complexity. However, despite few exceptions in which live cells or primitive organisms were encapsulated into a polymer matrix, no demonstration of an in vivo study case of scaffolds generated with the use of such a method was performed. Here, we report a preclinical study of 3D artificial microstructured scaffolds out of hybrid organic-inorganic (HOI) material SZ2080 fabricated using the DLW technique. The created 2.1 × 2.1 × 0.21 mm(3) membrane constructs are tested both in vitro by growing isolated allogeneic rabbit chondrocytes (Cho) and in vivo by implanting them into rabbit organisms for one, three and six months. An ex vivo histological examination shows that certain pore geometry and the pre-growing of Cho prior to implantation significantly improves the performance of the created 3D scaffolds. The achieved biocompatibility is comparable to the commercially available collagen membranes. The successful outcome of this study supports the idea that hexagonal-pore-shaped HOI microstructured scaffolds in combination with Cho seeding may be successfully implemented for cartilage tissue engineering.