Isolation and Characterization of Skin Derived Mesenchymal Stem Cell (SMSCs) from New Zealand Rabbit, Oryctolagus Cuniculus: A in Vitro Study

Mesenchymal stem cells (MSCs) exhibit high proliferation and self-renewal capabilities and are critical for tissue repair and regeneration. Furthermore, they can be isolated from a variety of tissues and have many potential applications in the clinical setting. In this study, Skin Derived Mesenchymal Stem Cells (SMSCs) were isolated from a New Zealand rabbit and analyzed for the skin sampling area, separation method, culture conditions, primary and passage culture times, and cell surface markers. The result shows that SMSCs have surface markers of mesenchymal stem cells by immunofluorescence of CD105, CD90, CD73, CD44, and CD200 and the CD105, CD90, and CD73 positive markers were also confirmed with Flow cytometry. Furthermore, negative markers of CD45 and CD34 did not show in immunofluorescence but CD45, CD34, and CD133 were confirmed with Flow cytometry

Osteogenic potential of gingival stromal progenitor cells cultured in platelet rich fibrin is predicted by core-binding factor subunit-α1/Sox9 expression ratio (in vitro) [version 1; referees: 1 approved, 2 approved with reservations]

Background: Alveolar bone defect regeneration has long been problematic in the field of dentistry. Gingival stromal progenitor cells (GSPCs) offer a promising solution for alveolar bone regeneration. In order to optimally differentiate and proliferate progenitor cells, growth factors (GFs) are required. Platelet rich fibrin (PRF) has many GFs and can be easily manufactured. Core-binding factor subunit-α1 (CBF-α1) constitutes a well-known osteogenic differentiation transcription factor in SPCs. Sox9, as a chondrogenic transcription factor, interacts and inhibits CBF-α1, but its precise role in direct in vitro osteogenesis remains unknown. GSPCs cultured in vitro in PRF to optimally stimulate osteogenic differentiation has been largely overlooked. The aim of this study was to analyze GSPCs cultured in PRF osteogenic differentiation predicted by CBF-α1/Sox9. Methods: This study used a true experimental with post-test only control group design and random sampling. GPSCs isolated from the lower gingiva of four healthy, 250-gram, 1-month old, male Wistar rats (Rattus Novergicus) were cultured for two weeks, passaged every 4-5 days. GSPCs in passage 3-5 were cultured in five M24 plates (N=108; n=6/group) for Day 7, Day 14, and Day 21 in three different mediums (control negative group: αModified Eagle Medium; control positive group: High Glucose-Dulbecco’s Modified Eagle Medium (DMEM-HG) + osteogenic medium; Treatment group: DMEM-HG + osteogenic medium + PRF). CBF-α1 and Sox9 were examined with ICC monoclonal antibody. A one-way ANOVA continued with Tukey HSD test (p0.05) was performed. Results: The treatment group showed the highest CBF-α1/Sox9 ratio (16.00±3.000/14.33±2.517) on Day 7, while the lowest CBF-α1/Sox9 ratio (3.33±1.528/3.67±1.155) occurred in the control negative group on Day 21, with significant difference between the groups (p<0.05). Conclusion: GSPCs cultured in PRF had potential osteogenic differentiation ability predicted by the CBF-α1/sox9 ratio

In vitro bone sialoprotein-I expression in combined gingival stromal cells and platelet rich fibrin during osteogenic differentiation

Purpose: To analyze the expression of bone sialoprotein - I (BSP - I) after the addition of platelet rich fibrin (PRF) in gingival somatic cell (GSC) culture medium during osteogenic differentiation in vitro. Methods: GSCs were extracted from healthy, 1-month-old, male Wistar rats (Rattus Novergicus), weighing 250 - 300 g, and which had been randomly selected (n=4). These cells were cultured for 14 days and passaged every 4 days. Five subcultures of GSCs were cultured in three plates (M24) (N = 54; n = 6) for 7, 14 and 21 days in three preconditioned culture media (group I: plain culture media; group II: preconditioned osteogenic culture media, and group III: preconditioned osteogenic culture media with platelet rich fibrin). The expression of BSP-I was immunocytochemically (ICC) examined with monoclonal antibodies. Homogeneity and normality tests (p > 0.05) were then performed followed by an analysis of variance (ANOVA, p < 0.05). Results: The highest expression of BSP-I was found in group III (Day 21, 13.00 ± 2.000), while the lowest expression of BSP-I was found in group I (Day 7, 7.33 ± 1.155). There were significant differences between the groups (p = 0.000, p < 0.05). Conclusion: PRF stimulates and significantly enhances the expression of BSP-I in GSC culture during osteogenic differentiation. Thus, PRF can be used to accelerate regeneration of alveolar bone defects

The potency of hematopoietic stem cells (hscs) and natural killer (nk) cells as a therapeutic of sars-cov-2 Indonesia isolates infection by viral inactivation (in vitro Study)

Background: The prevalence of COVID-19 cases in Indonesia as of June 9, 2020, has been confirmed 32.076 positive cases, with 1.923 death cases. The total number of deaths reached 92,941 cases. There has been a recent update on stem cell-based biological, medical therapy as an optional treatment to handling COVID-19 due to its potential viability besides using the prevalent conventional chemical drug therapy. Methods: In this study, in vitro research was conducted to determine the potential of hematopoietic stem cells (HSCs) and natural killer cells (NK cells) against SARS-CoV-2 viruses, which virus isolates were collected in Indonesia. The SARS-CoV-2 virus was planted in rat kidney cells and Vero cells. The cells that had been planted with the virus were given HSCs and NK cells, followed by being evaluated at intervals of 24, 48, and 72 hours. The evaluation was done by collecting cells and supernatant from the cell plate and then determining the viral load using a Polymerase Chain Reaction (PCR) machine. Results: The results showed that the addition of HSCs and NK on cells that had been infected by SARS-CoV-2 resulted in a decrease in viral load within 24 to 72 hours in all variations of Multiples of Infection (MoI) values. Conclusions: The administration of HSCs and NK cells has the potential to eliminate the SARS-CoV-2 virus. Although this study is only an in vitro study, it could be the basis for the development of alternative stem cell-based therapies to tackle COVID-19 cases

Gingival Mesenchymal Stem Cells from Wistar Rat’s Gingiva (Rattus Novergicus) – Isolation and Characterization (In Vitro Study)

Gingiva is emerging as a source of Mesenchymal Stem Cells. Gingival Mesenchymal Stem Cell has been isolated and characterized from the gingival connective tissue of wistar rat (Rattus Novergicus). Gingival Mesenchymal Stem Cell sources are rich, attainable and easy to isolate through minimal invasive procedure. Gingival Mesenchymal Stem Cells are ideal to accelerate bone regeneration. The aim of this study was to analyze Gingival Mesenchymal Stem Cells from Wistar Rats’ gingiva (Rattus Novergicus) isolation and characterization by CD34, CD44, CD73, CD90, CD105 expression. This study was descriptive observational with simple random sampling method. Gingival Mesenchymal Stem Cells were isolated from healthy, 200 gram, 1 month year old, male rat’s (Rattus Novergicus) lower gingival tissue through gingivectomy procedure (n=4). Gingiva were minced into small fragments then cultured in 2 weeks. The culture was passaged every 3-5 days after cultured and plated. The isolated Gingival Mesenchymal Stem Cells in passage 5 were characterized by CD34, CD44, CD73, CD90, CD105 using Immunocytochemistry and flowcytometry examination. Gingival Mesenchymal Stem Cells strongly expressed CD44+, CD73+, CD90+, CD105+ but did not express CD45- and CD34-. Gingival Mesenchymal Stem Cells’ morphology was fibroblast-like, spindle-shaped, colony-forming abilities, and stick to the culture plate. Gingiva is potential Stem Cell source. Gingival Mesenchymal Stem Cells has multipotency ability with proliferation and mesenchymal stem cells characteristic advantageous for tissue engineering and regenerative therapy

Potensi Aplikasi Kombinasi Platelet Rich Fibrin (PRF) Dengan Skin-Derived Mesenchymal Stem Cell (SMSCs) Pada Regenerasi Luka Kulit Full-Thickness Kelinci

Skin full-thickness wound in animals can be triggers to stress conditions and lead to poor quality of life. PRF and SMSCs have the potential in regeneration. Skin derived mesenchymal stem cells identification using ICC with antibody-labelled FITC CD105, CD73, CD90, CD45 then MTT assays conducted to see proliferation effect. Measurments of PDGF, VEGF, IGF, TGF-b were used ELISA. After 21 days, fibronectin and MMP8 in skin tissue was perfomed with IHC. The highest level of growth factor (PDGF, VEGF, IGF, TGF-b) was seen in the combination of PRF and SMSCs group. The SMSCs showed proliferation after combination with PRF. The fibronectin expression was highest in the PRF and SMSCs combination group, while MMP8 expression was seen in all groups. PRF and SMSCs had abundant of growth factors that helped in skin injury regeneration. It helped fibroblasts proliferation, migration and forming ECM (including fibronectin). Expression of MMP8 indicated tissue was in remodeling phase after 21 days. The combination of PRF and SMSCs shows better regeneration response in full-thickness skin wound

The Potency of Hematopoietic Stem Cells (HSCs) and Natural Killer (NK) Cells as A Therapeutic of SARS-CoV-2 Indonesia Isolates Infection by Viral Inactivation (In Vitro Study)

Background: The prevalence of COVID-19 cases in Indonesia as of June 9, 2020, has been confirmed 32.076 positive cases, with 1.923 death cases. The total number of deaths reached 92,941 cases. There has been a recent update on stem cell-based biological, medical therapy as an optional treatment to handling COVID-19 due to its potential viability besides using the prevalent conventional chemical drug therapy. Methods: In this study, in vitro research was conducted to determine the potential of hematopoietic stem cells (HSCs) and natural killer cells (NK cells) against SARS-CoV-2 viruses, which virus isolates were collected in Indonesia. The SARS-CoV-2 virus was planted in rat kidney cells and Vero cells. The cells that had been planted with the virus were given HSCs and NK cells, followed by being evaluated at intervals of 24, 48, and 72 hours. The evaluation was done by collecting cells and supernatant from the cell plate and then determining the viral load using a Polymerase Chain Reaction (PCR) machine. Results: The results showed that the addition of HSCs and NK on cells that had been infected by SARS-CoV-2 resulted in a decrease in viral load within 24 to 72 hours in all variations of Multiples of Infection (MoI) values. Conclusions: The administration of HSCs and NK cells has the potential to eliminate the SARS-CoV-2 virus. Although this study is only an in vitro study, it could be the basis for the development of alternative stem cell-based therapies to tackle COVID-19 cases

An in vitro study of dual drug combinations of anti-viral agents, antibiotics, and/or hydroxychloroquine against the SARS-CoV-2 virus isolated from hospitalized patients in Surabaya, Indonesia

A potent therapy for the infectious coronavirus disease COVID-19 is urgently required with, at the time of writing, research in this area still ongoing. This study aims to evaluate the in vitro anti-viral activities of combinations of certain commercially available drugs that have recently formed part of COVID-19 therapy. Dual combinatory drugs, namely; LopinavirRitonavir (LOPIRITO)-Clarithromycin (CLA), LOPIRITO-Azithromycin (AZI), LOPIRITODoxycycline (DOXY), Hydroxychloroquine (HCQ)-AZI, HCQ-DOXY, Favipiravir (FAVI)-AZI, HCQ-FAVI, and HCQ-LOPIRITO, were prepared. These drugs were mixed at specific ratios and evaluated for their safe use based on the cytotoxicity concentration (CC50) values of human umbilical cord mesenchymal stem cells. The anti-viral efficacy of these combinations in relation to Vero cells infected with SARS-CoV-2 virus isolated from a patient in Universitas Airlangga hospital, Surabaya, Indonesia and evaluated for IC50 24, 48, and 72 hours after viral inoculation was subsequently determined. Observation of the viral load in qRT-PCR was undertaken, the results of which indicated the absence of high levels of cytotoxicity in any samples and that dual combinatory drugs produced lower cytotoxicity than single drugs. In addition, these combinations demonstrated considerable effectiveness in reducing the copy number of the virus at 48 and 72 hours, while even at 24 hours, post-drug incubation resulted in low IC50 values. Most combination drugs reduced pro-inflammatory markers, i.e. IL-6 and TNF-α, while increasing the anti-inflammatory response of IL-10. According to these results, the descending order of effective dual combinatory drugs is one of LOPIRITO-AZI>LOPIRITO-DOXY>HCQ-AZI>HCQ FAVI>LOPIRITO-CLA>HCQ-DOX. It can be suggested that dual combinatory drugs, e.g. LOPIRITO-AZI, can potentially be used in the treatment of COVID-19 infectious diseases

A Randomized, Double-Blind, Multicenter Clinical Study Comparing the Efficacy and Safety of a Drug Combination of Lopinavir/Ritonavir-Azithromycin, Lopinavir/Ritonavir-Doxycycline, and Azithromycin-Hydroxychloroquine for Patients Diagnosed with Mild to Moderate COVID-19 Infections

At the present time, COVID-19 vaccines are at the testing stage, and an effective treatment for COVID-19 incorporating appropriate safety measures remains the most significant obstacle to be overcome. A strategic countermeasure is, therefore, urgently required. Aim. +is study aims to evaluate the efficacy and safety of a combination of lopinavir/ritonavirazithromycin, lopinavir/ritonavir-doxycycline, and azithromycin-hydroxychloroquine used to treat patients with mild to moderate COVID-19 infections. Setting and Design. +is study was conducted at four different clinical study sites in Indonesia. +e subjects gave informed consent for their participation and were confirmed as being COVID-19-positive by means of an RTPCR test. +e present study constituted a randomized, double-blind, and multicenter clinical study of patients diagnosed with mild to moderate COVID-19 infection. Materials and Methods. Six treatment groups participated in this study: a Control group Madministered with a 500 mg dose of azithromycin; Group A which received a 200/50 mg dose of lopinavir/ritonavir and 500 mg of azithromycin; Group B treated with a 200/50 mg dose of lopinavir/ritonavir and 200 mg of doxycycline; Group C administered with 200 mg of hydroxychloroquine and 500 mg of azithromycin; Group D which received a 400/100 mg dose of lopinavir/ritonavir and 500 mg of azithromycin; and Group E treated with a 400/100 mg dose of lopinavir/ritonavir and 200 mg of doxycycline. Results. 754 subjects participated in this study: 694 patients (92.4%) who presented mild symptoms and 57 patients (7.6%) classified as suffering from a moderate case of COVID-19. On the third day after treatment, 91.7%–99.2% of the subjects in Groups A–E were confirmed negative by a PCR swab test compared to 26.9% in the Control group. Observation of all groups which experienced a significant decrease in virus load between day 1 and day 7 was undertaken. Other markers, such as CRP and IL-6, were significantly lower in all treatment groups (p< 0.05 and p< 0.0001) than in the Control group. Furthermore, IL-10 and TNF-α levels were significantly elevated in all treatment groups (p< 0.0001). +e administration of azithromycin to the Control group increased CRP and IL-6 levels, while reduced IL-10 and TNF-α on day 7 (p< 0.0001) compared with day 1. Decreases in ALTand AST levels were observed in all groups (p< 0.0001). +ere was an increase in creatinine in the serum level of the Control, C, D, and E groups (p< 0.05), whereas the BUN level was elevated in all groups (p< 0.0001). Conclusions. +e study findings suggest that the administration of lopinavir/ritonavir-doxycycline, lopinavir/ritonavir-azithromycin, and azithromycinhydroxychloroquine as a dual drug combination produced a significantly rapid PCR conversion rate to negative in three-day treatment of mild to moderate COVID-19 cases. Further studies should involve observation of older patients with severe clinical symptoms in order to collate significant amounts of demographic data

The distribution pattern and growth factor level in platelet-rich fibrin incorporated skin-derived mesenchymal stem cells: An in vitro study

Background and Aim: A skin wound in an animal must be cared for to prevent further health issues. Platelet-rich fibrin (PRF) and skin-derived mesenchymal stem cells (SMSCs) have been reported to have potential in increasing the rate of wound healing. This study aimed to analyze the distribution patterns and levels of platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and transforming growth factor-β (TGF-β) in PRF incorporated with SMSCs. Materials and Methods: This study employed a true experiment (in vitro) design with post-test only performed in the control group alone. PRF and SMSCs were extracted from the blood and skin of 16 rabbits. SMSCs were characterized using immunocytochemistry to examine clusters of differentiation for 45, 73, 90, and 105. PRF was incorporated into the SMSCs and then divided into four groups (N=32/n=8): Group A (PRF only), Group B (PRF+SMSCs, incubated for 1 day), Group C (PRF+SMSCs, incubated for 3 days), and Group D (PRF+SMSCs, incubated for 5 days). Scanning electron microscopy was used to examine the distribution pattern of SMSCs between groups. The supernatant serum (Group A) and supernatant medium culture (Group D) were collected for the measurement of PDGF, IGF, VEGF, and TGF-β using an enzyme-linked immunosorbent assay sandwich kit. An unpaired t-test was conducted to analyze the differences between Groups A and D (p<0.01). Results: Group D had the most morphologically visible SMSCs attached to the PRF, with elongated and pseudopodia cells. There was a significant difference between the levels of growth factor in Groups A and D (p=0.0001; p<0.01). Conclusion: SMSCs were able to adhere to and distribute evenly on the surface of PRF after 5 days of incubation. The PRF incorporated SMSCs contained high levels of PDGF, IGF, VEGF, and TGF- β, which may prove to have potential in enhancing wound healing